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LIBRARY 
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A, 


A  MANUAL  OF   BACTERIOLOGY 


WILLIAMS 


A  MANUAL 


OF 


BACTERIOLOGY 


' 


HERBERT  U.  WILLIAMS,  M.D. 

PROFESSOR    OF.  PATHOLOGY    AND    BACTERIOLOGY,    MEDICAL     DEPARTMENT, 
UNIVERSITY    OF   BUFFALO 


WITH   NINETY-NINE  ILLUSTRATIONS 


THIRD   EDITION,   REVISED  AND   ENLARGED 


PHILADELPHIA : 
P.    BLAKISTON'S    SON  &    CO. 

1012  WALNUT   STREET 
1903 


Main  Lib. 
Depi. 


fee  LlMAftV  Of 
COM6NESS, 

T*»  C*pfcM  Itoctiwtf 

•CT  24  1903 

entry 


BUM 


BIOLOGY 

LIBRARY 

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Copyright,    1903 
By  HERBERT  U.  WILLIAMS. 


PRtSS  OF 

ERA  PRINTING  COMPANY, 
LANCASTER  PA, 


PREFACE    TO    THE    THIRD    EDITION. 

THE  plan  used  in  the  preceding  editions  of  this  manual 
has  been  followed  in  the  preparation  of  the  present  one. 
The  only  departures  have  been  in  the  insertion  of  a  short 
historical  sketch  and  the  freer  use  of  references  to  original 
articles  and  reviews.  It  is  hoped  that  these  features  will 
assist  in  arousing  the  interest  of  students.  As  far  as  pos- 
sible, reference  has  been  made  to  articles  in  American  and 
English  journals  likely  to  be  easy  of  access.  Besides  the 
ones  just  named,  numerous  additions  have  been  made  which 
the  recent  advances  in  our  knowledge  have  rendered  neces- 
sary. Most  of  the  illustrations  of  apparatus  are  new.  The 
photomicrographs  also  are  new  and  original  with  a  few 
exceptions  noted  in  the  text.  It  is  probably  needless  to  say 
that  none  of  them  were  retouched.  The  writer  is  indebted 
to  the  Gratwick  Laboratory  of  Buffalo  for  the  use  of  its 
facilities  in  making  these  photographs. 

BUFFALO,  NEW  YORK,  August,  1903. 


268453 


PREFACE    TO    THE    SECOND    EDITION. 

ALTHOUGH  there  has  been  no  lack  of  works  on  bacteri- 
ology, it  seemed  to  the  writer  that  there  was  still  a  field 
open  for  one  which  sought  to  give  the  portions  essential  to 
medical  science  in  a  concise  manner.  It  is  gratifying,  there- 
fore, that  the  first  edition  of  this  little  book  should  have  been 
exhausted  so  soon. 

Whether  wisely  or  not,  it  is  a  fact  that  many  medical 
schools  require  their  students  to  absorb  an  amount  of 
knowledge  that  taxes  the  brain  to  the  utmost.  While  such 
conditions  remain,  the  need  is  urgent  for  presenting  what 
is  taught  in  the  accessory  branches  in  as  condensed  a  form 
as  is  consistent  with  a  clear  understanding  of  their  great 
fundamental  principles.  It  is  mastery  of  such  principles, 
after  all,  which  is  the  object  of  a  course  in  bacteriology, 
for  they  are  essential  to  a  correct  understanding  of  most  of 
the  other  branches.  After  that  has  been  accomplished, 
(including  the  applications  of  bacteriology  to  diagnosis), 
it  must  be  admitted  that  other  branches  deserve  a  larger 
amount  of  the  student's  time.  This  may  be  said  without 
meaning  to  minimize  the  importance  of  bacteriology  in  the 
training  of  a  physician.  In  the  opinion  of  the  writer  it  is 
neither  possible  nor  desirable  that  every  graduate  should 
be  a  trained  bacteriologist.  However,  no  instructor  can 
hope  to  bring  the  principles  above  mentioned  home  to  his 
classes  except  by  laboratory  work.  Very  little  attempt 
has  been  made  to  outline  the  program  of  a  laboratory 
course,  as  that  will  always  need  to  be  planned  according 
to  the  circumstances  under  which  it  is  given. 


PREFACE.  Vll 

The  purpose  of  this  book  is  to  give  in  the  smallest  pos- 
sible space  the  facts  which  a  physician  must  know,  with 
some  of  those  which  it  is  desirable  that  he  should  know, 
and  a  little  of  that  which  he  may  learn  if  his  needs  or  in- 
clinations lead  him  to  go  further.  It  is  acknowledged, 
however,  that,  in  deference  to  precedent,  this  purpose  has 
not  been  carried  to  its  fullest  extent.  Much  time  has  been 
spent  on  the  index,  in  order  to  make  the  contents  quickly 
accessible.  It  is  a  source  of  regret  to  the  writer  that  the 
additions  which  the  revision  seemed  to  demand  have  made 
the  present  book  a  little  larger  than  the  first  edition. 

BUFFALO,  NEW  YORK,  June,  1901. 


PREFACE    TO    THE    FIRST    EDITION. 

IN  this  manual  the  writer  has  endeavored  to  describe 
the  laboratory  technique  which  the  beginner  must  follow, 
and  at  the  same  time  to  give  a  concise  summary  of  the 
facts  in  bacteriology  most  important  to  the  physician.  In 
preparing  a  work  of  this  character,  which  claims  to  be 
nothing  more  than  a  compilation,  the  standard  text-books 
were  necessarily  consulted  freely.  On  account  of  the  need 
for  brevity  it  has,  in  most  cases,  been  impossible  to  men- 
tion authorities. 

The  writer  is  glad  to  have  this  opportunity  to  acknowl- 
edge his  obligation  to  the  works  of  Sternberg,  Fliigge, 
Gunther,  Eisenberg,  Abbott,  W.  H.  Park,  Muir  and  Ritchie, 
Vaughan  and  Novy,  and  McFarland;  and  to  numerous 
papers  by  Professor  Welch  and  others.  It  is  thought  that 
the  chapters  on  Germicides,  and  Surgical  Disinfection,  by 
Drs.  Thos.  B.  Carpenter  and  Marshall  Clinton,  will  be  use- 
ful not  only  for  the  information  presented  in  them,  but 
especially  in  correlating  that  information  with  the  facts  of 
bacteriology. 

BUFFALO,  NEW  YORK,  October,  1898. 


Vlll 


CONTENTS. 


INTRODUCTION 1 1 

Historical  Sketch 18 

PART  I. 

BACTERIOLOGICAL  TECHNIQUE. 
CHAPTER   I. 

PAGE. 

Examination  of  Bacteria  with  the  Microscope,  including  Methods  of 
Staining    29 

CHAPTER    II. 
Sterilization  ur 

CHAPTER    III. 
Culture-media   71 

CHAPTER    IV. 

The  Cultivation  of  Bacteria.    Tube-cultures ;  the  Incubator ;  Anaer- 
obic   Methods 84 

CHAPTER    V. 

The  Cultivation  of  Bacteria    (Continued).     Isolation  of  Bacteria; 

Plate-cultures    0.6 

CHAPTER    VI. 
Inoculation  of  Animals.    Autopsies ;    Collodion  Sacs 103 

CHAPTER   VII. 
Collection    of    Material 108 

CHAPTER    VIII. 

Systematic  Study  of  Species  of  Bacteria.     Suggestions   for  Class- 
work;    Rules    112 

ix 


X  CONTENTS. 

PART  II. 

CHAPTER    I. 

Classification;    General  Morphology  and  Physiology  of  Bacteria.  ...   117 

CHAPTER    II. 
Products  of  the  Growth  of  Bacteria 128 

CHAPTER    III. 
Distribution  of  Bacteria — Soil,  Air,  Water,  Foods 133 

CHAPTER    IV. 
Bacteria  of  the  Normal  Human  Body 151 

CHAPTER    V. 
Bacteria   in   Disease 157 

CHAPTER    VI. 
Toxins     17.2 

CHAPTER    VII. 
Immunity    176 

CHAPTER    VIII. 
Disinfectants  and  Antiseptics.    By  Thomas  B.  Carpenter,  M.D 194 

CHAPTER    IX. 
Preparation  of  Instruments,  Ligatures,  Dressings,  etc.,  for  Surgical 

Purposes.     By  Marshall   Clinton,   M.D 211 

PART  III. 

NON-PATHOGENIC  BACTERIA 221 

Yeasts  and  Moulds 231 

PART  IV. 
PATHOGENIC   BACTERIA. 

Suppuration   and   Allied   Conditions 235 

Staphylococcus   pyogenes   aureus 243 

albus    245 

epidermidis   albus 246 

Streptococcus  pyogenes 246 

of  Erysipelas 250 

Micrococcus   tetragenus 250 

lanceolatus    (of    Pneumonia) 251 

melitensis 255 


CONTENTS.  xi 

Diplococcus  intracellularis  meningitidis 256 

Micrococcus  of  Gonorrhea 258 

Bacillus  of  Soft  Chancre 260 

pnetimoniae    (of    Friedlander)      260 

of    Rhinoscleroma 261 

pyocyaneus    262 

"       proteus    264 

of  Bubonic  Plague 265 

aerogenes   capsulatus 268 

of  Malignant  Edema 270 

of    Tetanus 270 

of  Anthrax 273 

"       of    Influenza 277 

of  Diphtheria 278 

"       tuberculosis   287 

of    Leprosy 296 

"       mallei  (of  Glanders) 297 

Actinomyces  bovis 299 

Bacillus  of  Typhoid  Fever 301 

coli   communis 309 

lactis  aerogenes 313 

of    Dysentery 313 

Spirillum  of  Asiatic  Cholera 315 

Spirilla  Allied  to  the  Spirillum  of  Asiatic  Cholera 323 

Spirillum   of   Relapsing   Fever 326 

APPENDIX. 

PATHOGENIC   PROTOZOA. 

Amoeba  of  dysentery 328 

Malarial   parasite 330 

Small-pox  and  Vaccinia 334 

Yellow   fever 335 

Trypanosomes   335 


LIST  OF  ILLUSTRATIONS. 


FIG.  PAGE. 

1.  Micrococci,  Bacilli,  Spirilla 14 

2.  Test-tube    with    Culture-medium 17 

3.  Microscope   30 

4.  Abbe    Condenser 31 

5.  Platinum    Wires 33 

6.  Hanging-drop     34 

7.  Cornet  Forceps  for  Cover-glasses 37 

8.  Stewart   Forceps   for   Cover-glasses 38 

9.  Kirkbride    Forceps    for    Slides 38 

10.  Schanze   Microtome <o 

11.  Hot-air   Sterilizer 62 

12.  Arnold    Steam    Sterilizer 64 

13.  Massachusetts  Board  of  Health  Sterilizer 65 

14.  Koch  Steam  Sterilizer 66 

15.  Autoclave    68 

1 6.  Kitasato    Filter 69 

17.  Test-tube  with  Potato 78 

1 8.  Wire   Basket   for   Test-tubes 82 

19.  Manner  of  Holding  Test-tubes 85 

20.  Stab-culture    86 

21.  Smear-culture 86 

22.  Incubator    88 

23.  Reichert  Gas-regulator 89 

24.  Gas-regulator    89 

25.  Koch    Automatic    Gas-burner 90 

26.  Buchner's   Method   for   Cultivating   Anaerobes 92 

27.  Frankel's                                                                        93 

28.  Novy's                                                                            94 

29.  Cover-glass  as  used  to  Distinguish  Anaerobes  from  Aerobes 95 

30.  Petri    Dish 98 

31.  Dilution-cultures  in  Esmarch  Roll-tubes facing  99 

32.  Appearance  of  Colonies  on  Gelatin  in  a  Petri  Dish facing  99 

33.  Esmarch's    Roll-tube 100 

34.  Mouse-holder   103 

35.  Apparatus  for  the  Subcutaneous  Insertion  of  Solid  Substances..  ..  104 

xiii 


XIV  ILLUSTRATIONS. 

36.  McCrae's  Method  for  making  Collodion  Capsules 106 

37.  Cover-glass   Preparation  of  Blood 100 

38.  Sternberg   Bulb no 

39.  Micrococci  of  Various  Forms 118 

40.  Bacilli   of  Various   Forms 119 

41.  Spirilla  of  Various  Forms 119 

42.  Involution     Forms 120 

43.  Bacteria    with    Capsules .* 122 

44.  Bacteria    with    Spores 122 

45.  Bacteria    Showing    Flagella 124 

46.  Fermentation-tube     131 

47.  Sedgwick-Tucker  Aerobioscope 136 

48.  Diagram  to  Illustrate  Side-chain  Theory  of  Immunity 185 

49.  Bacillus  subtilis 226 

50.  Spirilla  from   Swamp  Water 228 

51.  Spirilla   from   Swamp   Water   with    Flagella 229 

52.  Yeast  Cells 23 1 

53.  Penicillium    glaucum,    Oidium    lactis,    Aspergillus   glaucus,    Mucor 

mucedo    232 

54.  Staphylococcus  pyogenes  aureus  in  Pus 242 

55.  Pure    Culture 243 

56.  Culture  in   Gelatin 245 

57.  Streptococcus  pyogenes,  Pure  Culture 247 

58.  in    Pus 248 

59.  "         Culture    on    Agar 249 

60.  Micrococcus  tetragenus  in  Pus 250 

61.  lanceolatus  (of  Pneumonia)  in  Sputum 252 

62.  lanceolatus    (of   Pneumonia)    showing   Capsules 253 

63.  Diplococcus  intracellularis  meningitidis 257 

64.  Gonococcus  in  Pus 259 

65.  Bacillus    pyocyaneus 263 

66.  Bacillus  of  Bubonic  Plague 265 

67.  Bacillus  aerogenes   capsulatus 268 

68.  Culture 269 

69.  "       of  Tetanus 271 

70.  "       of  Anthrax 273 

71.  with    Spores 274 

72.  "       Colony   275 

73.  "       Culture    275 

74.  "       showing  Concave  Ends 276 

75.  "       in  the  Liver 277 

76.  "       of    Diphtheria 279 

77.  Neisser's    Stain 280 


ILLUSTRATIONS.  XV 

78.  Tubes   for   Cultivation  of  Diphtheria   Bacillus 281 

79.  Bacillus    of    Diphtheria,    Culture 282 

80.  "       tuberculosis     287 

81.  Branching  Form  of  Tubercle  Bacillus 288 

82.  Bacillus  tuberculosis,   stained,   in    Sputum 289 

83.  Ray- fungus  of  Actinomycosis,  Fresh  Preparation 299 

84.  Actinomyces  bovis  from  a  Pure  Culture 300 

85.  Bacillus   of  Typhoid   Fever 302 

86.  "  "  "       with     Flagella 303 

87.  Widal  Serum-reaction  with  Typhoid  Bacilli 305 

88.  Bacillus  coli  communis 310 

89.  with  Flagella 311 

90.  Spirillum  of  Cholera 316 

91.  Involution   Forms 317 

92.  "         Colonies   on    Gelatin   plates 318 

93.  "         Culture  in   Gelatin 319 

94.  Vibrio    proteus 324 

95.  Spirillum  of  Relapsing  Fever 327 

96.  Malarial   Parasite 331 

97-  "  "       33i 

98.  "        33i 

99-  "        331 


INTRODUCTION. 


ANYONE  who  has  not  himself  worked  in  a  bacteriological 
laboratory  finds  it  difficult  to  form  a  vivid  conception  of 
what  bacteria  are  like,  because  among  the  familiar  animals 
and  plants  there  are  none  with  which  a  close  comparison 
can  be  made.  Of  the  common  organisms,  perhaps  ordi- 
nary yeasts  and  moulds  are  most  like  the  bacteria.  Yeasts 
and  moulds,  as  everyone  knows,  grow  on  bread,  cheese, 
meat,  syrups  and  the  like.  They  flourish  in  moist  and 
dark  places,  as  do  mushrooms,  puffballs  and  the  other 
fungi.  All  these  fungi,  appearing  so  different  in  some  re- 
spects, are  alike  in  one  particular,  which  is  the  absence  of 
the  green  color  that  we  are  apt  to  think  of  as  being  the 
essential  feature  of  vegetation.  Plants  that  are  green  owe 
their  color  to  a  substance  called  chlorophyll.  Upon  the 
properties  of  this  substance  one  of  the  most  fundamental 
facts  in  biology  depends.  Under  the  influence  of  sunlight, 
by  means  of  chlorophyll,  plants  are  able  to  use  as  food  the 
carbon  dioxide  which  is  always  present  in  the  atmosphere 
in  small  amounts.  Although  carbon  dioxide  is  one  of  the 
most  simple  and  stable  of  compounds,  the  union  of  its  com- 
ponent elements  is  broken  by  the  plant,  and  they  are  em- 
ployed in  the  formation  of  other  much  more  complex  and 
unstable  compounds,  such  as  starch  and  cellulose,  which 
enter  into  the  plant's  structure.  The  work  of  plants,  it  will 
be  noticed,  is,  in  the  main,  precisely  the  reverse  of  that  per- 
formed by  animals.  Animals  take  the  unstable  carbohy- 
drates wTith  high  potential  energy,  such  as  starches  and 


12  MANUAL    OF    BACTERIOLOGY. 

sugars,  as  food,  and  exhale  the  stable  carbon  dioxide  from 
the  lungs.  At  the  same  time  the  animal  receives  the  bene- 
fit of  the  energy  resulting  from  the  oxidation  of  the  carbo- 
hydrates, which  may  appear  indirectly  in  the  form  of  nerv- 
ous or  muscular  activity  or  warmth. 

Those  plants  that  are  devoid  of  chlorophyll  are  compelled 
to  some  extent  to  use  the  same  kinds  of  food  as  animals. 
They  are  unable  to  decompose  carbon  dioxide  (in  most 
cases),  and  procure  their  nourishment  from  the  dead  or 
living  bodies  of  other  plants  or  animals.  Since  they  have 
no  chlorophyll,  light  is  of  no  advantage  to  them,  and  is 
often  a  positive  detriment.  Bacteria  contain  no  chlorophyll, 
and  are  usually  classed  with  the  fungi,  which  they  resem- 
ble in  their  inability  to  decompose  carbon  dioxide  and  to 
use  it  as  food.1 

There  is  another  well-known  property,  possessed  by 
yeasts  especially,  which  may  be  useful  in  explaining  the 
work  done  by  bacteria.  It  is  a  fact  of  every-day  observa- 
tion that,  when  yeasts  grow  in  dilute  solutions  of  sugar, 
alcohol  and  gas  are  formed.  It  not  only  appears  that 
bacteria  sometimes  form  alcohol  and  gas  from  sugar,  but 
that  with  different  kinds  of  bacteria  and  different  kinds  of 
food  material  a  great  number  of  substances  are  made,  some 
of  which  are  powerful  poisons.  In  most  of  the  diseases 
caused  by  bacteria  such  poisons  are  produced  within  the 
living  body  of  the  patient.  The  symptoms  of  the  disease 
and  the  changes  in  the  patient's  body  are  due  to  these 
poisons,  rather  than  to  the  direct  action  of  bacteria. 

The  extreme  smallness  of  the  bacteria  prevents  us  from 
seeing  them  as  individuals  without  the  aid  of  the  micro- 
scope, although  great  numbers  of  them  taken  together  may 
form  a  plainly  visible  mass  or  growth.  When  they  are 
examined  with  the  microscope  they  appear  as  little  round, 

'See  Chapter  I,  Part  II. 


INTRODUCTION.  13 

rod-shaped  or  curved  bodies,  which  may  be  likened  to  so 
many  periods,  dashes  and  commas.  It  is  at  once  perceived 
that  each  bacterium  is  an  individual  by  itself,  and  that  it 
consists  of  a  single  cell,  not  of  an  aggregation  of  cells,  as 
do  most  of  the  common  plants  and  animals. 

Under  favorable  conditions  bacteria  may  be  seen  to  mul- 
tiply, one  organism  being  divided  by  a  partition  into  two 
parts,  which  separate  and  become  two  new  organisms. 
The  process  is  called  fission. 

At  times  certain  bacteria  present  little  bright  spots  which 
enlarge,  and  from  which  the  rest  of  the  cell  breaks  away 
in  fragments.  The  bright  body  that  remains  is  called  a 
spore,  and  has  greater  resisting  power  against  injurious 
influences  than  has  the  fully  developed  organism.  To  this 
extent  these  spores  are  something  like  the  seeds  of  higher 
plants.  There  are  spores  that  can  withstand  boiling  for 
hours,  but  fortunately  that  is  not  true,  as  far  as  we  know, 
of  the  spores  of  any  of  the  bacteria  that  produce  disease. 
The  earlier  investigators  observed  the  appearance  of  bac- 
teria in  nutrient  infusions  which  they  had  endeavored  to 
sterilize  by  heat.  They  looked  upon  this  fact  as  indicating 
the  possibility  of  spontaneous  generation,  and  it  furnished 
the  chief  support  of  that  theory.  Probably  their  fluids 
contained  very  resistant  spores,  and  were  in  reality  not 
sterile. 

From  these  facts,  a  definition  for  bacteria  may  be  formu- 
lated. 

Bacteria  (Greek  paxTijpeov,  meaning  a  little  stick)  are  ex- 
tremely minute,  unicellular  plants,  which  have  no  chloro- 
phyll, and  zvhich  divide  by  fission.  They  are  sometimes 
called  schizomycetes.  In  every-day  language  they  are 
known  as  microbes,  and  also  as  germs.  They  are  gener- 
ally classed  with  the  fungi.  In  some  respects  they  seem 
quite  closely  related  to  the  algae  or  simplest  green  plants, 


14  MANUAL    OF    BACTERIOLOGY. 

and,  on  the  other  hand,  they  have  strong  points  of  likeness 
with  some  of  the  unicellular  animals  belonging  to  the  in- 
fusoria. 

Bacteria  are  divided  into  three  great  groups : 

Micrococci,  or  cocci1  (singular,  coccus) — spherical  forms. 

Bacilli  (sing.,  bacillus) — long  and  straight,  or  rod-shaped 
bacteria. 

Spirilla  (sing.,  spirillum) — consisting  of  spiral  filaments 
like  the  turns  of  a  corkscrew,  or  parts  of  spirals  shaped 
like  commas. 

The  extreme  smallness  of  the  bacteria  is  hard  of  com- 
prehension. We  may  say,  of  most  of  them,  that  from 
5,000  to  25,000  placed  end  to  end  would  make  a  line  about 

an  inch  in  length.  When  one 
touches  a  growth  of  bacteria 

K?    ^£^\l      "/NT/"          with  the  sterilized  platinum  wire 
•  •  ••  v  \  .j* 

'  ^  %  ^  (j  r  j        anc{    spreads    the    tiny    portion 

Micrococci.  Bacilli.  Spirilla.       that  adheres  to  the  wire  upon  a 

slip  of  glass,  it  is  found  upon  ex- 
amination with  the  microscope  that  the  bacteria  left  on 
the  glass  may  be  compared  to  the  stars  in  the  sky,  the 
grains  of  sand  on  the  shore,  or  any  of  the  other  standards 
for  numbers  that  are  nearly  beyond  computation. 

It  is  well  known  that  bacteria  are  present  on  most  of  the 
objects  about  us.  They  occur  on  the  skins  of  men  and 
other  animals,  as  well  as  in  the  mouth,  stomach  and  intes- 
tines, and  on  most  of  the  surfaces  of  the  body  that  open 
to  the  external  w^orld.  They  are  found  in  the  water  of 
rivers  and  lakes,  and  in  the  ocean.  They  appear  in  the 
soil  down  to  a  depth  of  several  feet.  They  float  in  the 
air,  except  at  high  altitudes  and  over  the  ocean.  Nansen 

1  Pronounced  kok-si  or  kok-ke :  see  Webster's  International,  Century, 
and  Standard  Dictionaries,  and  Foster's  and  Keating's  Medical  Diction- 
aries. The  writer  knows  of  no  authority  for  the  prevailing  pronuncia- 
tion kok-kl. 


INTRODUCTION.  1 5 

found  bacteria  on  the  ice  of  the  Polar  sea.  Investigators 
have  even  reported  finding  them  fossilized,  indicating,  as 
we  might  expect,  that  they  existed  at  remote  periods  in  the 
earth's  history.  But  the  vast  majority  of  them  are  entirely 
harmless  as  far  as  we  are  concerned,  and  many  of  them 
are  indispensable  in  maintaining  the  balance  existing  be- 
tween the  different  kinds  of  living  things. 

Were  it  not  for  the  putrefactive  and  nitrifying  bacteria 
the  dead  bodies  of  plants  and  animals  would  lie  practically 
unchanged  where  they  fell,  and  the  fertilization  of  the  soil 
necessary  for  the  life  of  most  plants,  by  means  of  substances 
derived  from  such  dead  material,  would  cease. 

In  northern  Siberia  the  bodies  of  the  extinct  species  of 
elephant  called  mammoths  have  been  found  imbedded  in 
frozen  soil  where  they  appear  to  have  lain  for  thousands  of 
years.  In  this  case  the  growth  of  putrefactive  bacteria  has 
been  prevented  by  cold,  as  in  the  modern  refrigerator  or 
cold-storage  plant. 

Some  bacteria  have  been  made  to  do  work  in  industries, 
like  the  bacilli  whose  growth  in  cream  imparts  an  agree- 
able flavor  to  the  butter  and  cheese. 

Bacteria  are  also  made  use  of  in  the  manufacture  of 
vinegar. 

The  study  of  bacteria  has  led  to  the  understanding  of 
many  hitherto  unexplained  facts.  The  unaccountable  de- 
velopment of  a  moist,  brilliant  red  deposit  on  bread  and 
other  articles  of  food,  which  was  formerly  believed  by  the 
superstitious  to  be  blood,  deposited  by  some  miraculous 
agency,  we  know  to  be  due  to  the  growth  of  a  common  or- 
ganism (bacillus  prodigiosus).  The  emission  of  light  by 
decaying  substances  when  seen  in  the  dark  may  be  caused 
by  bacteria  as  well  as  other  organisms. 

It  seems  that  in  some  cases  in  which  death  was  attributed 
to  the  suction  of  air  into  the  veins,  because  air  appeared  to 


1 6  MANUAL    OF    BACTERIOLOGY. 

be  present  inside  the  heart,  the  air  was  in  reality  a  gas, 
formed  by  certain  bacilli  that  invaded  the  body  just  before 
or  just  after  death  (bacillus  aerogenes  capsulatus). 

Woodhead  tells  us  that  some  savages  are  in  the  habit  of 
smearing  the  soil  of  certain  localities  upon  their  arrows  for 
an  arrow-poison,  which  is  intelligible  in  the  light  of  the  fact 
that  soil  often  contains  the  bacilli  of  tetanus  (lockjaw). 

The  comparatively  small  number  of  species  of  bacteria 
that  cause  disease  are  the  ones  that  interest  us  most,  and 
are  those  which  have  been  most  carefully  studied.  The 
necessity  that  falls  upon  bacteria,  in  common  with  other 
fungi,  to  derive  their  food  from  organic  matter  makes  it 
easy  to  understand  that  they  should  frequently  exist  as  para- 
sites upon  living  animals  and  plants.  Pear-blight  and  some 
other  diseases  of  plants  are  caused  by  bacteria.  We  find 
that  frogs,  birds,  cattle  and  a  great  number  of  animals 
besides  men  suffer  from  diseases  produced  by  bacteria. 

When  bacteria  are  placed  upon  slips  of  glass  they  may  be 
studied  with  the  microscope  while  alive.  Some  of  them 
when  living  are  motionless ;  others  wriggle  vigorously. 
Some  dart  about  like  minnows  in  a  stream,  or  they  make 
their  way  slowly  across  the  field  of  the  microscope  like  a 
boat  that  is  being  sculled  from  the  stern.  By  proper  methods 
it  can  be  shown  that  the  movements  are  effected  through 
one  or  more  fine,  hair-processes,  called  flagella. 

Often  it  is  expedient  to  study  bacteria  after  drying  them 
on  slips  of  glass,  when  they  may  be  made  more  conspicuous 
by  giving  them  an  artificial  color  (staining).  Some  of  the 
substances  of  which  they  are  composed  readily  absorb  cer- 
tain dyes.  For  this  purpose  the  aniline  dyes  are  used,  and 
their  employment  has  been  one  of  the  important  factors  in 
making  progress  in  bacteriology  possible. 

With  the  microscope  alone  it  is  not  usually  practicable  to 
distinguish  accurately  between  various  kinds  of  bacteria. 


INTRODUCTION. 


Micrococci,   for  instance,  which  are,  in  reality,   extremely 
different,  may  look  very  much  alike.     The  differences  are 
usually  apparent  when  the  bacteria  are  grown  artificially. 
The  cultivation  is  done  for  the  most  part  in        FIG.  2. 
test-tubes  containing  some  material  which  fur-       s^"y 
nishes  suitable  food.     The  nutrient  materials     c£ 

£'•':•••' '-•;,.    .,.  rf£*. 

most  used  are  meat-extract  and  peptone,  which, 
dissolved  with  salt  in  water,  constitute  nutrient 
bouillon.  Ordinary  gelatin,  or  a  vegetable  gel- 
atin called  agar-agar ,  may  be  added  to  the 
bouillon  when  a  solid  culture-medium  is  de- 
sired. Before  these  substances  can  be  used  for 
the  cultivation  of  bacteria  all  other  bacteria 
which  they  might  contain  must  be  destroyed  by 
heat. 

When  bacteria  are  to  be  conveyed  from  one 
tube  to  another,  or  from  a  tube  to  a  glass  slide, 
in  order  to  examine  them  with  the  microscope, 
the  manipulation  is  performed  on  a  platinum 
wire  fastened  into  a  glass  rod.  The  rules  laid 
down  for  the  management  of  the  tubes  and  the 
platinum  wire  (Part  I.,  Chapter  VIII.)  must  ~  . 

Test-tube     con- 
be  carefully  followed.    There  is  little  or  no  dan-     taining  cui- 

ger  in  bacteriological  work  if  the  proper  pre- 
cautions are  conscientiously  observed;  but  carelessness  may 
lead  to  disastrous  and  even  fatal  results,  as  has  happened 
more  than  once. 

Finally,  the  effects  of  bacteria  in  bringing  about  disease 
may  be  tested  on  the  lower  animals.  The  proof  that  a  par- 
ticular species  of  bacteria  causes  a  particular  disease  cannot 
be  considered  complete  unless  the  disease  can  be  repro- 
duced by  introducing  these  bacteria  into  some  animal. 

The  student  who  wishes  to  pursue  bacteriological  study 
in  any  direction  farther  than  it  is  possible  for  the  limits  of 


1 8  MANUAL    OF    BACTERIOLOGY. 

a  short  manual  to  go,  may,  besides  consulting  the  large 
text-books,  and  weekly  medical  journals,  obtain  much 
assistance  from  technical  periodicals.  The  Journal  of  Ex- 
perimental Medicine,  Journal  of  Medical  Research,  and  the 
Journal  of  Applied  Microscopy,  published  in  this  country, 
and  the  English  Journal  of  Pathology  and  Bacteriology  and 
Journal  of  Hygiene  will  give  a  great  deal  that  is  valuable. 

A  reading  knowledge  of  German  and  French  is  very 
desirable.  The  Centralblatt  fiir  Bakteriologie,  etc.,  a 
German  weekly,  and  the  Bulletin  de  I'Institut  Pasteur, 
published  bimonthly  in  Paris,  contain  abstracts  of  most  of 
the  important  researches  made  in  all  parts  of  the  world. 
The  Annalcs  de  I'Institut  Pasteur,  the  Zeitschrift  fiir  Hy- 
giene, and  the  Archiv  fiir  Hygiene  contain  many  original 
articles  on  bacteriological  subjects. 

The  whole  literature  of  any  specified  subject  in  bacteri- 
ology can  be  most  conveniently  found  in  Baiungarten's 
Jahresbericht  dcr  Mikrodrganisinenlclire. 

Historical  Sketch. — The  remarkable  growth  of  mechan- 
ical and  industrial  enterprises  which  the  last  half  century 
has  witnessed  is  held  to  be  characteristic  of  it.  The  world 
justly  takes  pride  in  its  achievements  along  these  lines. 
Nearly  all  that  we  know  of  bacteria  and  the  part  they  play  in 
producing  disease  has  been  learned  during  the  same  period. 
It  is  but  fair  to  say  that  the  rapid  growth  of  this  knowledge 
has  been  equally  characteristic  of  the  age. 

Nevertheless  many  facts  were  known  long  ago,  and  even 
by  the  ancients,  which  were  effective  in  directing  the  thought 
of  later  years.  The  epidemic  nature  of  certain  maladies 
was  naturally  among  the  earliest  of  these  to  be  noticed,  and 
was,  even  until  recently,  attributed  to  the  influence  of  gods, 
demons,  or  other  supernatural  agencies.  The  superstitions 
and  crude  beliefs  of  the  past  gave  rise  to  a  mass  of  grotesque 


INTRODUCTION.  1 9 

theories  and  fanciful  speculations.  But  with  all  this  we 
hear  of  certain  beliefs  and  practices  which  plainly  fore- 
shadowed those  of  the  present  day.  Latin  writers  nearly 
two  thousand  years  ago  recorded  a  relation  between  insects 
and  malaria,  which  has  but  lately  been  proved  and  ex- 
plained. The  treatment  of  lepers  by  the  Hebrews  resembles 
that  now  in  vogue :  "  He  is  unclean :  he  shall  dwell  alone ; 
without  the  camp  shall  his  habitation  be  "  (Lev.  XIII.  46). 
There  is,  in  fact,  much  in  the  laws  of  Moses  that  points  to 
some  knowledge  of  the  nature  of  infections.  '  This  is  the 
law,  when  a  man  dieth  in  a  tent :  all  that  come  into  the  tent 
and  all  that  is  in  the  tent  shall  be  unclean  for  seven  days. 
And  every  open  vessel  which  has  no  covering  upon  it  shall 
be  unclean"  (Numb.  XIX.  14,  15). 

"  Everything  that  may  abide  the  fire,  ye  shall  make  it  go 
through  the  fire,  and  it  shall  be  clean  "  (Numb.  XXXI.  23). 

In  Homer  we  read  of  Ulysses,  that,  having  slain  his 
wife's  troublesome  suitors : 

"  With  fire  and  sulphur,  cure  of  noxious  fumes, 

He  purged  the  walls  and  blood-polluted  rooms"  (Pope's  Odyssey). 

The  massive  aqueducts  of  the  Romans  still  remain  to 
testify  that  they  understood  the  importance  of  a  pure  water- 
supply. 

In  Rome  there  were  also  sewers  for  the  disposal  of  drain- 
age; while  the  Cretans  and  Assyrians  used  sewerage  sys- 
tems hundreds  and  even  thousands  of  years  before. 

About  the  fourteenth  century  we  find  quarantine  against 
infectious  diseases,  plague  in  particular,  practiced  by  certain 
Italian  cities ;  and  the  word  "  quarantine  "  came  into  use 
from  the  fact  that  the  period  of  detention  was  about  forty 
days  (Ital.  quarantine*)  * 

1  The  Early  History  of  Quarantine,  J.  M.  Eager,   Yellow  Fever  hist. 
Bui.,  No.  12,  U.  S.  Marine  Hosp.  Service. 


20  MANUAL    OF    BACTERIOLOGY. 

Leeuwenhoek,  a  citizen  of  Delft,  in  Holland  (1632- 
1723),  appears  to  have  been  the  first  who  actually  saw  bac- 
teria. Yeast-cells  he  certainly  observed,  besides  making 
many  other  contributions  of  great  value  to  biology.  Leeu- 
wenhoek produced  admirable  lenses  of  high  magnifying 
power,  and  described  what  he  witnessed  with  singular  accu- 
racy and  enthusiasm. 

Even  before  this  time  men  had  sought  to  explain  the 
phenomena  of  infectious  diseases  by  supposing  the  body 
to  have  been  penetrated  by  minute  parasites,  for  example 
worms.  The  spread  of  such  diseases  through  a  community 
from  a  single  center  could  readily  be  accounted  for  by  the 
multiplication  of  a  contagious  element,  itself  alive  (con- 
taghnn  vivum).  With  increasing  knowledge  of  the  abund- 
ance of  microscopic  life  these  speculations  took  firmer  hold. 
But  long  before  their  truth  was  finally  demonstrated  great 
advances  \vere  made  in  the  prevention  of  infectious  diseases. 
Much  honor  is  due  the  clinicians  whose  accurate  observa- 
tions and  foresight  accomplished  important  results  at  an 
early  day,  working  with  what  now  seems  a  very  meagre 
knowledge  of  the  facts. 

The  production  of  immunity  against  small-pox  by  inocu- 
lation was  first  practiced  in  oriental  countries.  The  method 
had  long  been  in  use  in  the  East,  when  in  1718  it  was 
brought  to  the  notice  of  Europeans  by  Lady  Montagu,  wife 
of  the  English  ambassador  at  Constantinople.  The  proced- 
ure consisted  simply  of  the  introduction  of  the  virus  of 
small-pox  by  puncture  of  the  skin.  An  attack  of  small-pox 
resulted,  which  was  much  milder  and  far  less  dangerous 
than  the  natural  disease. 

Lady  Montagu  stated  in  a  letter :  "  Every  year  thousands 
undergo  the  operation;  and  the  French  ambassador  says 
pleasantly  that  they  take  the  small-pox  here  by  way  of 
diversion,  as  they  take  the  waters  in  other  countries."  The 


INTRODUCTION.  21 

mild  attacks  that  followed  inoculation  were,  however,  just 
as  contagious  to  other  persons  as  the  natural  disease,  so  that 
the  dangers  of  this  practice  to  the  community  were  very 
great. 

A  much  better  method  was  found  in  vaccination.  At  this 
time  a  belief  was  current  among  farmers  that  a  mild  form 
of  disease,  called  cow-pox,  acquired  by  milkers,  furnished 
protection  against  small-pox.  This  belief  was  investigated 
and  introduced  to  the  world  by  Edward  Jenner.  In  1796 
he  inoculated  his  first  patient  with  cow-pox.  In  a  few  years 
the  practice  of  vaccination  spread  to  all  parts  of  the  world.1 

It  was  introduced  into  the  United  States  by  Dr.  Benjamin 
Waterhouse  of  Harvard.  President  Thomas  Jefferson  was 
active  in  bringing  it  into  general  use  especially  in  the  south. 

As  early  as  1847  Semmelweis  of  Vienna  attributed  the 
origin  of  puerperal  fever  to  poisons  carried  by  the  fingers  of 
physicians  and  students,  whose  hands  had  been  soiled  in 
the  dissecting  room.  To  this  he  was  led  by  the  death  of  a 
friend  from  pyemia  following  a  dissection-wound.  He 
noted  the  similarity  of  the  course  of  his  friend's  case  with 
cases  of  puerperal  fever.  He  advocated  washing  the  hands 
of  the  attendant  in  solutions  of  chlorin  or  chlorid  of  lime, 
in  addition  to  cleansing  them  with  soap  and  water. 

The  cause  of  puerperal  fever  was  still  unknown.  En- 
deavors to  connect  it  with  atmospheric  influences  and  the 
like  had  been  unsuccessful.  During  the  seventeenth  and 
eighteenth  centuries  it  had  been  attributed  to  the  absorption 
of  milk  into  the  blood  from  the  breasts.  Semmelweis  stood 
his  ground  in  spite  of  opposition  and  ridicule,  though  he 
somewhat  modified  his  doctrine.  His  views  agree  substan- 
tially with  the  practice  of  the  present  day  which  they  have 
greatly  influenced. 

1  See  the  works  of  Edward  Jenner  by  Dock,  N.  Y.  Med.  Jour.,  Nov. 
29  and  Dec.  6,  1902 ;  also  The  History  of  Vaccination,  by  Dulles,  Phila- 
delphia Medical  Journal,  May  30,  1903. 


22  MANUAL    OF    BACTERIOLOGY. 

During  the  same  period  similar  ideas  were  ad- 
vanced by  Dr.  Oliver  Wendell  Holmes  in  the  United  States. 
His  paper  on  "  The  Contagiousness  of  Puerperal  Fever  " 
appeared  in  1843.  A  lively  controversy  lasting  several 
years  was  provoked,  in  which  Holmes  defended  his  position 
with  great  vigor.  His  admirable  literary  style  served  him 
effectively.1 

In  the  first  half  of  the  nineteenth  century,  with  improved 
microscopes,  knowledge  of  minute  living  things  grew 
rapidly,  chiefly  with  respect  to  infusoria  and  other  relatively 
large  forms.  In  1840  Henle  described  the  part  played  by 
microorganisms  in  producing  disease  in  terms  surprisingly 
in  accord  with  views  held  at  the  present  time.  His  deduc- 
tions were  based  almost  entirely  on  knowledge  of  the  gen- 
eral nature,  spread  and  course  of  infections.  So  too, 
Villemin  anticipated  the  discovery  of  the  bacillus  of  tuber- 
culosis, for  he  transmitted  the  disease  to  animals,  by  inocu- 
lating them  with  material  from  cases  of  tuberculosis  in 
man. 

The  key  to  exact  knowledge  of  the  microorganisms  of 
disease  was  finally  discovered  in  the  study  of  fermentation. 
No  better  illustration  could  be  found  of  the  possible  value  to 
mankind  which  may  lie  in  any  addition  whatever  to  the 
common  stock  of  facts.  The  study  of  bottles  of  bad-smell- 
ing broth  would  have  seemed,  fifty  years  ago,  a  most  un- 
promising beginning  for  the  discovery  of  the  causes  of 
cholera,  plague,  and  the  like,  or  for  an  antitoxin  for  diph- 
theria. 

Studies  on  Fermentation  and  Spontaneous  Generation. — 
Two  observers  (Schwann,  Cagniard-Latour,  1837)  almost 
simultaneously  stated  the  proposition  that  yeast  cells  were 
living  organisms,  and  that  the  fermentation  of  solutions  of 
sugar  was  clue  to  their  growth.  From  this  time  ensued 

1  See  Medical  Essays,  O.  W.  Holmes,  Houghton,  Mifflin  &  Co.,  1889. 


INTRODUCTION.  23 

a  controversy  which  lasted  more  than  thirty  years.  The 
agency  ascribed  to  yeasts  was  energetically  denied  by  many, 
prominent  among  them  Liebig;  while  it  was  sustained  with 
vigor  by  others.  The  latter  extended  the  original  conception 
to  include  other  sorts  of  fermentation  and  the  putrefaction 
of  albuminous  material.  Different  kinds  of  fermentation, 
with  different  products,  such  as  acetic  acid  and  butyric  acid, 
were  ascribed  to  the  growth  of  different  kinds  of  microbes. 

These  microbes  were  found  to  be  fungi  of  various  sorts, 
and  chiefly  one  or  another  variety  of  bacteria.  The  most 
celebrated  among  the  students  of  fermentation  was  Pasteur, 
the  simplicity  and  kindliness  of  whose  character  excite  our 
admiration  equally  with  his  devotion  to  his  work.1 

Before  the  nature  of  fermentation  was  understood  the 
possibility  of  spontaneous  generation  had  been  universally 
admitted.  When  vermin  of  various  sorts  appeared  in  putre- 
fying material  the  conclusion  was  drawn  that  they  had  their 
origin  directly  from  it.  Although  that  had  long  since  been 
disproved  in  the  case  of  large  organisms  like  worms  and 
frogs,  still,  as  late  as  the  middle  of  the  last  century,  it  was 
held  by  many  to  account  for  the  swarming  microscopic  life 
found  in  fermenting  fluids.  A  flask  of  meat  broth  left  ex- 
posed to  the  air  will  after  a  few  days  contain  countless  tiny 
living  things,  chiefly  bacteria.  Pasteur  and  his  supporters 
showed  that  these  bacteria  were  the  progeny  of  others 
already  in  the  flask  or  which  had  fallen  in  from  the  air. 

When  the  flask  of  broth  was  boiled,  no  development  of 
organisms  took  place,  if  the  entrance  of  germs  from  the 
atmosphere  was  prevented.  The  latter  was  accomplished 
by  such  devices  as  heating  the  air,  passing  it  through  sul- 
phuric acid,  using  a  flask  with  a  long  twisted  neck  or  by 
plugging  the  flask  with  cotton  (Schroder  and  Von  Dusch). 

1  See  Louis  Pasteur,  His  Life  and  Labors  by  His  Son-in-Law,  trans- 
lated by  Lady  Claude  Hamilton. 


24  MANUAL    OF    BACTERIOLOGY. 

To  prove  that  boiling  had  not  made  the  fluid  unfit  for  the 
growth  of  organisms,  air  was  subsequently  allowed  to  have 
access  to  it  without  such  precautions,  when  putrefaction 
took  place  in  the  usual  manner. 

At  the  same  time  it  was  demonstrated  not  only  that  bac- 
teria are  present  in  all  fermenting  and  putrefying  sub- 
stances, but  that  they  exist  wherever  there  is  animal  life  or 
vegetation. 

These  principles  underlie  the  methods  used  daily  for  the 
preservation  of  meat,  fruit  and  vegetables,  in  the  household 
and  in  factories. 

Although  even  boiling  occasionally  failed  to  prevent  fer- 
mentation, investigators  came  with  practice  to  have  a 
smaller  number  of  failures.  Such  failures  it  was  shown 
were  due  to  the  presence  of  the  resistant  state  called  spores, 
which  some  bacteria  assume.  The  true  nature  of  spores 
was  recognized  later  by  Colin.  Pasteur  found  that  exposure 
to  temperatures  above  the  boiling  point  (no°C.)  would 
destroy  the  most  resistant  microbes  and  their  spores. 

The  controversies  over  fermentation  and  putrefaction 
lasted  almost  until  the  present  day.  They  were  productive 
of  numerous  benefits  to  the  arts  and  manufactures.  But 
what  is  of  more  importance  to  our  subject,  they  led  to  a 
vastly  better  understanding  of  all  kinds  of  microorganisms. 
The  study  of  bacteria  was  now  pursued  with  great  vigor. 
In  the  space  of  about  twenty-five  years,  most  of  what  we 
know  concerning  the  bacteria  of  disease  has  been  learned. 
The  period  of  rapid  progress  is  not  yet  completed.  Nearly 
every  year  yields  some  advance  of  great  importance. 

The  discussions  concerning  fermentation  and  putrefac- 
tion, were  still  going  on  when  Lister  made  his  brilliant 
deduction  that  suppuration  and  septic  processes  in  wounds 
were  a  species  of  fermentation  (1867).  From  this  came  the 
antiseptic  and  aseptic  methods  of  operating  and  of  dressing 


INTRODUCTION.  25 

wounds,  which  have  made  possible  the  wonderful  results  of 
modern  operative  surgery.1 

In  1834  the  parasite  of  itch  (an  insect,  Acarus  scabei)  was 
discovered,  and  the  cause  of  one  contagious  malady  deter- 
mined. 

Quite  early  in  the  nineteenth  century  also  the  relatively 
large  fungi  of  thrush  and  some  of  the  parasitic  skin  diseases 
were  discovered.  The  bacilli  of  anthrax,  which  are  also 
large,  were  seen  in  the  blood  of  animals  by  Pollender  in  1855 
and  Davaine  in  1863. 

Davaine  produced  anthrax  in  animals  by  injecting  into 
them  blood  containing  anthrax  bacilli.  But  complete  proof 
that  these  bacilli  were  the  cause  of  the  disease,  required  that 
they  should  produce  it  when  injected  alone  and  when  freed 
from  the  smallest  trace  of  material  derived  from  the  first 
diseased  animal.  Unless  these  conditions  were  complied 
with,  some  other  material,  for  example  an  enzyme  or  fer- 
ment, might  be  supposed  to  be  carried  from  the  first  to  the 
second  animal  and  to  be  the  real  cause  of  the  disease.  For 
this  purpose  it  was  necessary  to  cultivate  the  bacilli  in 
nutrient  fluids,  such  as  meat  broth,  as  was  done  by  Pasteur. 
It  then  became  possible  to  demonstrate  that  their  properties 
could  remain  unaltered  after  being  grown  in  successive 
generations  on  different  lots  of  broth.  As  bacteria  of  two 
or  three  species  were  often  encountered  in  mixtures,  it  be- 
came most  important  to  secure  a  method  by  which  the  differ- 
ent species  could  be  separated  from  one  another  and  be 
propagated  as  separate  "  pure  cultures."  This  was  done 
successfully  by  diluting  such  mixtures  greatly,  so  that  a 
drop  planted  in  a  new  tube  of  broth  should  contain  only  a 
single  organism.  The  growth  ensuing  would  of  course  con- 
sist of  the  same  kind  of  organism  exclusively.  Such  pro- 
cedures were  uncertain  and  very  laborious. 

1  See  History  of  Medicine,  Dr.  Roswell  Park. 


2O  MANUAL    OF    BACTERIOLOGY. 

Koch  introduced  in  1881  his  method  of  separating  bacteria 
by  "plating"  (described  in  Part  I.),  probably  the  most 
important  single  contribution  to  bacteriological  technique. 
He  also  brought  solid  culture-media  into  general  use  by  em- 
ploying gelatin.  Other  important  technical  improvements 
of  the  same  period  were  the  adoption  of  the  illuminating 
apparatus  of  Abbe  and  immersion  objectives,  and  of  aniline 
dyes  for  staining  bacteria  and  making  them  visible  ( Weigert 
and  Ehrlich).  Beginning  with  the  bacillus  tuberculosis  de- 
scribed by  Koch  in  1882,  a  large  number  of  pathogenic  bac- 
teria were  discovered  during  the  ensuing  years  in  rapid  suc- 
cession. 

The  application  of  the  newly-gained  knowledge  concern- 
ing the  bacteria  causing  infectious  diseases  to  the  preven- 
tion and  cure  of  these  diseases  was  begun  almost  imme- 
diately by  Pasteur.  A  few  facts  existed  to  guide  the  direc- 
tion of  the  research.  It  had  been  known  even  in  ancient 
times  that  one  attack  of  an  infectious  disease,  such  as  scarlet 
fever,  may  confer  immunity  from  subsequent  attacks. 

The  protection  against  small-pox  which  was  furnished  by 
vaccination  also  was  suggestive,  although  the  mechanism  by 
which  this  protection  came  about  was  not  understood. 

Pasteur  worked  on  the  theory  that  immunity  to  a  disease 
might  be  secured  by  producing  a  mild  attack  of  the  disease. 
Such  a  mild  attack  might  be  expected  to  follow  if  a  sus- 
ceptible individual  were  inoculated  with  microbes  of  lowered 
virulence.  Various  methods  were  employed  to  reduce  the 
virulence  of  bacteria,  notably  cultivation  at  high  tempera- 
tures (43°C.).  In  this  manner  Pasteur  was  able  to  produce 
immunity  against  a  number  of  the  diseases  of  the  lower 
animals.  His  method  of  inoculating  sheep  and  cattle  against 
anthrax  is  widely  and  successfully  used.  A  similar  prin- 
ciple has  led  to  the  preparation  of  a  vaccine  for  the  disease 
of  cattle  called  "  black  leg,"  and  such  vaccine  is  now  dis- 


INTRODUCTION.  27 

tributed  gratuitously  to  farmers  by  the  United  States  gov- 
ernment. Inoculation  of  human  subjects  with  the  atten- 
uated living  virus  of  a  disease  is  used  only  for  hydrophobia. 
This  method  also  was  invented  by  Pasteur. 

The  preparations  of  antitoxins  for  infectious  diseases 
(see  the  chapter  on  Immunity)  we  owe  to  Behring.  This 
portion  of  our  subject  belongs  entirely  to  the  present  day, 
and  is  now  being  studied  with  great  energy. 

Allusion  has  already  been  made  to  moulds  and  other 
microscopic  parasites  whose  nature  makes  their  study  almost 
inseparable  from  that  of  the  bacteria.  In  this  class  also 
belong  the  primitive  forms  of  animal  life  (Protozoa)  which 
are  the  causes  of  amebic  dysentery  (Losch,  1875)  and 
malaria  (Laveran,  1880).  The  disease  of  cattle  called 
"  Texas  fever  "  is  also  caused  by  a  protozoon.  Theobald 
Smith  in  the  United  States  discovered  that  the  parasite  of 
Texas  fever  is  conveyed  from  one  animal  to  another  by  an 
insect,  the  cattle-tick.  Since  then  it  has  been  shown  (by 
Manson,  Ross  and  others)  that  malaria  is  conveyed  from  a 
person  having  the  disease  to  one  not  affected  by  means  of 
mosquitoes.  It  now  appears  probable  that  a  similar  rela- 
tion exists  between  mosquitoes  and  yellow  fever.  The  part 
played  by  flies  and  other  insects  in  carrying  disease  germs  is 
still  receiving  active  attention  and  the  future  may  have  larger 
possibilities  in  store. 

It  is  encouraging  to  reflect  that  the  progress  of  bacte- 
riology has  been  made  by  gradual  and  logical  steps.  The 
great  discoveries  have  not  been  lucky  accidents,  but  have 
been  worked  out  patiently  and  with  deliberation. 


PART  I. 


CHAPTER   I. 

EXAMINATION    OF    BACTERIA    WITH     THE    MICROSCOPE,     IN- 
CLUDING   METHODS    OF    STAINING. 

The  Microscope. — The  microscope  consists  of  a  tubular 
body  which  carries  the  optical  parts,  and  which  can  be 
raised  or  lowered  for  focusing.  The  objectives  should  be 
three  in  number,  and  should  be  attached  to  the  body  by 
means  of  a  triple  nose-piece,  which  permits  any  objective 
to  be  turned  into  the  optical  axis  at  will.  The  eye-piece 
slips  into  the  upper  and  opposite  end  of  the  body  or  tube. 
The  arrangements  for  focusing  consist  of  a  rack  and  pinion 
which  accomplish  the  coarse  adjustment,  and  a  more  deli- 
cate fine  adjustment.  The  stage,  upon  which  the  objects 
to  be  examined  are  placed,  has  an  opening  in  the  middle. 
In  this  opening  an  iris  diaphragm  and  Abbe  condenser  are 
inserted.  The  iris  diaphragm  enables  one  to  alter  the  size 
of  the  opening  as  desired.  Beneath  the  stage  is  a  mov- 
able mirror,  of  which  one  side  is  plane  and  the  other  con- 
cave. All  of  these  parts  are  supported  on  a  short,  heavy 
pillar  which  is  fixed  in  the  horseshoe-shaped  base. 

The  essential  parts  of  the  microscope  are,  of  course,  the 
eye-piece  (German,  Ocular),  and  the  objective.  Objectives 
are  given  various  names  by  different  makers,  for  instance, 
A,  B,  C,  etc.,  or  i,  2,  3,  etc.;  or  they  are  named  according 
to  their  focal  distances,  as  f  inch,  J  inch,  -J  inch,  etc.  In 
bacteriological  work  a  rather  "  low  power  "  §  or  J  inch 

29 


3O  MANUAL    OF    BACTERIOLOGY. 

objective,  an  ordinary  "  high  power  "  |  to  ^  inch  dry  objec- 
tive, and  a  high  power  ^  inch  oil-immersion  objective  are 
needed.  The  magnification  with  the  f  or  J  inch  objective 


FIG. 


Microscope. 

is  about  75  to  100  diameters;  with  the  ]  to  ,1  inch  300  to 
500  diameters;  with  the  ^  immersion  750  to  1,000  diame- 
ters. The  magnification  varies  according  to  the  eye-piece 
used,  as  well  as  with  the  objective.  A  i  inch  and  1.1  inch 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     3! 

eye-piece  (Zeiss  No.  2  and  No.  4)  serve  well  for  most  pur- 
poses. The  eye-pieces  are  usually  named  arbitrarily,  like 
the  objectives.  The  oil-immersion  objective  is  used  in  the 
examination  of  bacteria  where  a  very  high  power  is  desired. 
A  layer  of  thickened  oil  of  cedar-wood  is  placed  between  the 
lower  surface  of  the  objective  and  the  upper  surface  of  the 
glass  covering  the  object  under  examination.  The  oil  must 
be  wiped  away  from  the  surface  of  the  objective  when  the 
examination  is  finished.  For  this  purpose  the  soft  paper 
sold  by  dealers  in  microscopical  apparatus  serves  admi- 
rably. Care  must  be  taken  not  to  scratch  the  lower  surface 
of  this  objective.  Oil  of  cedar-wood  furnishes  a  medium 

FIG.  4. 


Abbe  Condenser.      On  the  right  side  the  figure  gives  a  sectional  view. 

having  nearly  the  same  refractive  index  as  the  glass  of  the 
lens  and  the  glass  on  which  the  object  is  mounted,  and  it 
obviates  the  dispersion  of  light  which  takes  place  when  a 
layer  of  air  is  interposed  between  the  objective  and  the 
object,  as  happens  with  the  ordinary  dry  lens.  This  ob- 
jective is  used  in  connection  with  the  Abbe  condenser, 
which  consists  of  two  or  three  lenses  combined  so  as  to 
focus  the  rays  coming  from  the  plane  mirror  upon  the 
object.  The  condenser  gives  a  very  intense  illumination 
over  a  very  small  field.  The  condenser  is  not  necessary 
excepting  with  the  oil-immersion  objective.  If  it  is  used 
with  the  other  objectives  the  illumination  must  be  regulated 
by  lowering  the  condenser,  closing  the  diaphragm  more  or 


32  MANUAL    OF    BACTERIOLOGY. 

less,  and  substituting  the  concave  for  the  plane  mirror. 
It  is  to  be  remembered  that  more  depends  upon  securing  a 
distinct  picture  than  upon  a  very  high  magnification  of  the 
object. 

The  microscope  should  be  placed  in  front  of  the  observer 
on  a  firm  table.  The  observer  should  be  able  to  bring  the 
eye  easily  over  the  eye-piece  when  the  tube  of  the  micro- 
scope is  in  vertical  position.  Daylight  should  be  em- 
ployed if  possible.  When  artificial  illumination  is  neces- 
sary, an  ordinary  lamp,  a  Welsbach  burner  or  an  incan- 
descent electric  light  may  be  used.  It  is  best  to  modify  the 
artificial  light  by  inserting  a  sheet  of  blue  glass  between 
the  light  and  the  mirror. 

In  order  to  focus  upon  any  object,  having  first  secured  a 
satisfactory  illumination  with  the  mirror,  it  is  best,  begin- 
ning with  the  low  power  and  using  the  coarse  adjustment 
for  focusing,  to  bring  the  objective  quite  close  to  the  object, 
and  then,  with  the  eye  in  position,  to  raise  the  tube  until 
the  object  comes  into  focus.  The  exact  focusing  is  done 
with  the  fine  adjustment.  The  observer  should  keep  both 
eyes  open  when  using  the  microscope,  and  should  be  able 
to  use  either  eye  at  will. 

All  measurements  of  microscopic  objects  are  expressed 
in  terms  of  a  micromillimeter.  This  is  one-thousandth  of 
a  millimeter  (.001  mm.),  which  is  about  .2-t  (1MM)  of  an  inch. 
It  is  generally  called  a  micron  for  short,  and  is  denoted  by 
the  Greek  letter  ft.  For  example,  5  IJL  ==  .005  mm.  ==  -  (]lQ-$ 
inch. 

The  Preparation  of  Specimens  of  Bacteria  for  Exami- 
nation with  the  Microscope. — The  substance  under  ex- 
amination is  usually  placed  upon  thin  slips  of  glass  called 
cover-glasses.  The  material  is  spread  over  the  cover-glass 
by  means  of  a  platinum  wire  which  has  been  fixed  in  a 
glass  rod  about  six  inches  long.  Such  a  platinum  wire  is 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     33 

used  constantly  in  doing  bacteriological  work.  It  is  the 
tool  by  means  of  which  one  is  able  to  handle  bacteria  with 
impunity.  It  serves  in  fact  as  a  kind  of  additional  finger. 
The  platinum  wire  must  be  stiff  enough  not  to  bend  too 
easily,  and  yet  it  should  not  be  so  large  that  it  will  not  cool 
rapidly  after  heating.  A  good  size  for  most  purposes  is 
number  23,  American  wire  gauge  (Brown  and  Sharp). 
The  wire  may  be  straight  throughout  its  length,  or  the  tip 
may  be  bent  to  form  a  loop  (German,  Oese).  It  is  well 
to  follow,  from  the  beginning,  certain  rules  which  make 
the  use  of  the  platinum  wire  safe  and  accurate.  Every 
time  it  is  taken  into  the  hand  and  before  using  it  for  any 
manipulation  heat  it  in  the  flame  of  a  Bunsen  burner  or 


Straight  platinum  wire  and  platinum  wire  loop. 

an  alcohol  lamp  to  a  red  heat ;  and  always,  after  using  and 
before  putting  it  down,  heat  it  again  to  a  red  heat.  After 
the  needle  has  become  wet  by  clipping  it  in  a  fluid  and  is  to 
be  sterilized  in  the  flame,  it  is  necessary  to  avoid  "  sputter- 
ing "  of  the  fluid  by  bringing  the  wet  needle  gradually  to 
the  flame,  so  as  to  dry  the  material  adhering  to  it  before 
burning  it.  This  procedure  must  be  done  with  great  care 
when  the  wire  has  been  dipped  in  milk  or  other  substances 
containing  oil.  When  the  needle  "  sputters,"  as  it  is  called, 
from  too  rapid  heating,  particles  that  have  not  yet  been 
sterilized  may  be  thrown  some  distance.  On  no  account 
should  the  needle  touch  any  object  other  than  that  which 


34  MANUAL    OF    BACTERIOLOGY. 

it  is  intended  it  should  touch.  With  such  a  platinum  wire, 
which  has  been  properly  sterilized,  one  can  easily  remove 
portions  from  a  culture  of  bacteria,  or  from  a  fluid  in 
which  bacteria  are  supposed  to  be  present.  The  glass  rod 
in  which  the  platinum  wire  is  fixed  should  be  held  between 
the  thumb  and  forefinger  of  the  right  hand  like  a  pen.  (For 
the  manner  of  holding  test-tubes,  see  page  84.) 

The  Hanging-drop. — Living  bacteria  may  be  studied 
with  the  microscope  while  suspended  in  some  fluid  sub- 
stance. The  needle  having  been  heated  to  a  red  heat  in 
the  flame  and  having  been  allowed  to  cool,  a  small  portion 
of  the  culture  or  other  material  may  be  removed  with  it 
and  deposited  in  the  center  of  an  ordinary  cover-glass. 
The  needle  should  again  be  sterilized  in  the  flame.  When 

FIG.  6. 


Diagram  of  the  hanging-drop. 

cultures  on  solid  media  arc  to  be  examined,  a  small  particle 
may  be  mixed  with  a  drop  of  sterilized  water  or  bouillon. 
The  cover-glass  should  have  been  carefully  cleaned  and 
sterilized  over  the  flame.  The  cover-glass  with  the  small 
drop  of  fluid  material  held  in  sterilized  forceps  is  now  to  be 
inverted  over  a  sterilized  glass  slide,  which  has  a  concavity 
ground  in  the  middle  of  it.  Around  the  concavity,  the  slide 
should  be  smeared  with  vaseline.  In  this  manner  a  small 
air-tight  chamber  is  made.  This  slide  and  cover-glass  may 
be  put  upon  the  stage  of  the  microscope.  A  good  dry  lens, 
if  of  sufficiently  high  power,  is  more  convenient  for  ex- 
amining the  hanging-drop  than  an  oil-immersion.  If  the 
latter  be  used,  having  placed  a  drop  of  cedar-oil  on  the 
center  of  the  cover-glass,  and  a  good  light  having  been 
secured,  the  oil-immersion  objective  should  be  brought 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     35 

down  upon  this  drop  of  oil.  The  beginner  often  experi- 
ences difficulty  in  focusing  upon  a  hanging-drop.  It  is 
well  to  shut  off  most  of  the  light  by  means  of  the  iris  dia- 
phragm. Often  it  is  well  to  secure  the  focus  roughly  upon 
the  extreme  outer  edge  of  the  chamber,  or  to  find  the  edge 
of  the  drop  of  fluid  with  the  low  power  and  then  to  focus 
upon  this  edge  with  the  oil-immersion  objective.  Above  all 
things  guard  against  breaking  the  cover-glass  by  forcing 
the  objective  down  upon  it.  The  motility  of  certain  bacteria 
is  one  of  the  most  striking  phenomena  to  be  observed  in 
the  hanging-drop.  It  is  not  to  be  confused  with  the  so- 
called  "  Brownian  movement  "  which  is  exhibited  by  fine 
particles  suspended  in  a  watery  fluid.  It  is  well  for  the 
beginner  to  observe  the  character  of  the  Brownian  move- 
ment by  rubbing  up  some  carmine  in  a  little  water,  and  with 
the  microscope  to  study  the  trembling  motion  exhibited  by 
these  particles  of  carmine.  It  will  be  noticed  that,  although 
the  particles  oscillate,  no  progress  in  any  direction  is  ac- 
complished unless  there  are  currents  in  the  fluid.  Such 
currents  might  give  rise  to  the  impression  that  certain 
bacteria  possessed  motility  when  they  were,  in  fact,  power- 
less to  move  of  themselves.  In  the  hanging-drop  the 
multiplication  of  bacteria  can  be  studied,  the  formation  of 
spores  and  the  development  of  spores  into  fully  formed 
bacteria.  The  hanging-drop  has  recently  been  put  into 
service  for  the  demonstration  of  the  so-called  serum- 
reaction  with  the  bacillus  of  typhoid  fever.  Sometimes 
bacteria  must  be  watched  in  the  hanging-drop  for  hours, 
or  even  days,  and  it  may  be  necessary  to  keep  it  at  the 
temperature  of  the  human  body  for  this  length  of  time. 
Various  complicated  kinds  of  apparatus  have  been  devised 
for  this  purpose,  but  they  are  needful  only  with  special 
kinds  of  work.  When  the  hanging-drop  preparation  is  no 
longer  required,  the  slide  and  cover-glass  should  be 
4 


36  MANUAL    OF    BACTERIOLOGY. 

dropped  into  a  5  per  cent,  carbolic  acid  solution  and  after- 
ward sterilized  by  steam. 

Hanging-block  preparations,  which  \vere  introduced  by 
Hill,1  make  use  of  a  cube  of  nutrient  agar  instead  of  a  drop 
of  fluid.  Bacteria  are  distributed  on  the  surface  of  the  agar, 
which  is  then  applied  to  a  cover-glass,  and  mounted  like  a 
hanging-drop.  The  bacteria  are  kept  in  a  layer  close  to  the 
glass,  where  growth  may  be  studied. 

Cover-glass  Preparations. — The  study  of  bacteria  with 
the  microscope  is  for  the  most  part  done  by  means  of  smears 
made  upon  thin  slips  of  glass.  Such  slips  of  glass  are 
generally  called  cover-glasses.  It  is  best  to  obtain  the 
kind  sold  by  dealers  as  No.  i,  f  inch  squares. 

The  cover-glass  may  be  cleaned  best  by  immersion  in  a 
mixture  of  sulphuric  acid  and  bichromate  of  potassium  so- 
lution, and  afterward  washed  thoroughly  in  distilled  water, 
and  finally  in  alcohol.  A  stock  of  clean  cover-glasses  may 
be  kept  in  a  bottle  of  alcohol. 

CLEANING  FLUID. 

Potassium    bichromate 40  grams. 

Water    150  c.c. 

Dissolve  the  bichromate  of  potassium  in  the 
water,  with  heat ;  allow  it  to  cool ;  then  add 
slowly  and  with  care  sulphuric  acid,  com- 
mercial    230  c.c. 

For  most  purposes  it  is  sufficient  to  wash  the  cover-glass 
in  alcohol  containing  3  per  cent,  of  hydrochloric  acid.  It 
should  then  be  wiped  clean  with  a  piece  of  linen  cloth. 
"Whenever  it  is  taken  into  the  ringers  it  should  be  held  by  the 
edges,  never  by  the  flat  surfaces.  As  far  as  possible  it 
should  be  handled  with  the  forceps.  It  can  be  used  very 
conveniently  in  the  form  of  forceps  known  as  the  Cornet 
forceps,  or  in  the  modification  devised  by  Stewart.  Bac- 

1  Journal  of  Medical  Research,  Vol.  VII.,  March,  1902. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     37 

teria  may  be  placed  upon  the  cover-glass  by  allowing  the 
glass  to  fall  upon  one  of  the  colonies  of  bacteria,  on  a  gelatin 
or  agar  plate  (see  page  99),  which  will  adhere  to  it  in  part, 
producing  an  "  impression  preparation  "  (German,  Klatsch- 
preparat}.  Such  a  preparation,  after  drying  in  the  air,  is 
to  be  fixed  by  passing  it  through  the  flame  three  times.  (See 
below.)  The  forceps  with  which  it  is  handled  should  be 
sterilized  in  the  flame. 

Generally  bacteria  contained  in  fluids,  like  sputum,  or 
taken  from  the  surface  of  a  culture,  are  smeared  over  the 
cover-glass  by  means  of  the  platinum  wire  or  loop,  which 
must  be  heated  to  a  red  heat  before  and  after  the  opera- 

FIG.  7. 


Cornet  forceps  for  cover-glasses. 

tion.  Such  preparations  are  called  smear,  cover-glass, 
cover-slip,  or  film  preparations.  When  the  material  to  be 
spread  is  thick  or  very  viscid,  a  small  drop  of  distilled 
water  must  first  be  placed  in  the  center  of  the  cover-glass 
so  as  to  dilute  it.  Beginners  generally  take  too  much  ma- 
terial on  the  wire.  As  thin  a  smear  as  possible  is  made. 
It  is  allowed  to  dry  in  the  air;  this  should  occupy  a  few 
seconds.  The  drying  may  be  hastened  by  holding  the 
forceps  with  the  cover-glass  a  long  distance  above  the 
flame,  at  a  point  where  the  heat  would  cause  no  discomfort 
to  the  hand.  Having  dried  the  preparation,  it  is  to  be 
passed  through  the  flame  of  a  Bunsen  burner  or  alcohol 
lamp  three  times,  taking  about  one  second  for  each  transit. 
The  heat  of  the  flame  serves  to  dry  the  bacteria  upon  the 
cover-glass  and  fix  them  permanently  in  position;  it  is  not 


3«  MANUAL    OF    BACTERIOLOGY. 

sufficient,  however,  when  applied  in  this  manner,  to  kill  all 
kinds  of  bacteria,  especially  those  containing  spores.  After 
it  has  been  passed  through  the  flame  three  times  the 
preparation  may  be  stained  with  one  of  the  aniline  dyes, 
and  after  washing  in  water  and  drying  may  be  mounted, 

FIG.  8. 


_D 


Stewart  forceps  for  cover-glasses. 

face  down,  in  Canada  balsam  upon  a  glass  slide.  It  makes 
a  suitable  object  to  be  examined  with  the  oil-immersion 
objective.  The  slide  is  a  thin  slip  of  glass,  3  inches  by  i 
inch,  with  ground  edges. 

The  smear  preparation  may  equally  well  be  made  directly 
upon  the  glass  slide.  The  fixation  in  the  flame  must  then 
occupy  a  longer  time  than  with  the  small  and  thin  cover- 

FIG.  9. 


Kirkbride   forceps   for  holding  slides. 

glass.  Such  preparations  have  the  advantage  that  several 
may  be  made  upon  one  slide,  and  that  after  staining  them 
they"  may  be  examined  in  cedar-oil,  with  the  oil-immersion 
lens,  without  the  use  of  the  cover-glass  and  Canada  balsam. 
The  forceps  of  Kirkbride  will  be  found  convenient  when 
staining  on  the  slide.  Experiments  performed  in  the 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     39 

writer's  laboratory  have  shown  that  the  ordinary  method 
of  fixation  in  the  flame,  when  applied  to  bacteria  spread 
upon  slides,  has  little  effect  on  the  vitality  of  many  species. 
The  beginner  is,  therefore,  advised  to  make  his  preparations 
on  cover-glasses. 

When  very  resistant  or  dangerous  pathogenic  bacteria 
are  being  handled,  after  fixation  by  heat  upon  the  slide  or 
cover-glass,  the  preparation  may,  if  desired,  be  immersed  in 
i-iooo  solution  of  bichloride  of  mercury  long  enough  to 
kill  the  bacteria,  without  injuring  the  preparation  or  its 
staining  properties. 

Staining. — The  staining  of  bacteria  is  done  for  the  most 
part  with  the  aniline  dyes.  The  object  of  staining  bacteria 
is  to  give  them  artificially  some  color  which  makes  them 
distinct  and  easily  visible  without  imparting  this  color  to 
the  substance  or  medium  in  which  they  are  imbedded.  The 
substances  known  as  aniline  dyes  are  derivatives  of  coal-tar, 
but  not  always  of  aniline.  These  dyes  are  of  great  import- 
ance in  bacteriological  work.  Their  number  is  very  large, 
but  only  a  few  are  in  common  use.  It  is  important  to  have 
the  purest,  and  those  manufactured  by  Griibler  are  reliable. 

It  is  simplest  to  classify  the  aniline  dyes  as  acid  or  basic. 
Eosin,  picric  acid  and  acid  fuchsin  are  acid  dyes;  they  tend 
to  stain  tissues  diffusely.  Fuchsin,  gentian-violet  and 
methylene-blue  are  basic  dyes ;  they  have  an  affinity  for  the 
nuclei  of  tissues  and  for  bacteria;  they  therefore  are  the 
dyes  used  chiefly  in  bacteriological  work.  The  other  va- 
rieties may  be  employed  as  contrast-stains;  another  con- 
trast-stain frequently  used  is  Bismarck  brown.  It  is  best 
to  keep  on  hand  saturated  solutions  of  the  aniline  dyes  in 
alcohol,  from  which  watery  solutions  may  be  made  when 
needed  by  adding  a  few  drops  of  the  alcoholic  solution  to 
a  small  dish  filled  with  water.  The  alcoholic  solution  is 
diluted  about  ten  times,  or  so  as  to  make  a  liquid  which  is 


40  MANUAL    OF    BACTERIOLOGY. 

just  transparent  in  a  layer  about  12  mm.  in  thickness,  after 
filtering. 

Fuchsin  and  gentian-violet  operate  rapidly  and  intensely. 
Methylene-blue  works  more  slowly  and  feebly;  it  is  to  be 
preferred  where  the  bacteria  occur  in  thick  or  viscid  sub- 
stances, like  pus,  mucus,  and  milk. 

Method  of  Staining  Cover-glass  Preparations. — (a)  A 
smear  preparation  of  bacteria  having  been  made  in  the 
manner  above  described,  and  a  watery  solution  of  either 
fuchsin,  gentian-violet  or  methylene-blue  having  been  pre- 
pared, the  cover-glass  is  to  be  dropped  into  a  dish  contain- 
ing the  dye,  or  the  dye  may  be  dropped  upon  the  cover- 
glass  held  in  the  forceps. 

(fr)   Allow  the  stain  to  act  for  about  thirty  seconds. 

(c)  Wash  in  water. 

(d)  Examine  with  the  microscope  in  water  directly  or 
after  drying  and  mounting  in  Canada  balsam. 

The  rapidity  and  intensity  of  staining  may  be  increased 
by  warming  the  solution  slightly.  The  bacteria  will  usually 
appear  more  distinct  if,  directly  after  pouring  off  the  stain, 
the  preparation  is  rinsed  for  a  few  seconds  in  i  per  cent, 
solution  of  acetic  acid,  and  then  thoroughly  washed  in  water. 
The  acetic  acid  solution  serves  to  remove  in  a  measure  any 
color  which  has  been  imparted  to  the  background,  and  which 
is  undesirable. 

Preparations  that  are  mounted  at  first  in  water  may  be 
made  permanent  by  moistening  the  edge  of  the  cover-glass 
so  that  it  may  easily  be  removed  from  the  slide,  then  dry- 
ing and  mounting  in  Canada  balsam.  Cover-glass  prepara- 
tions which  have  been  stained  are  examined  with  the  oil- 
immersion  objective,  employing  the  plane  mirror,  having 
the  iris  diaphragm  open  and  the  condenser  close  to  the 
lower  surface  of  the  glass  slide.  The  purpose  is  to  obtain 
the  most  intense  illumination  possible  over  a  small  field. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     4! 

The  watery  solutions  of  aniline  dyes  prepared  as  above  de- 
scribed deteriorate  in  a  short  time,  and  it  is  best  to  prepare 
them  freshly  each  time  they  are  required.  A  very  useful 
solution,  which  is  permanent,  is  Loffler's  alkaline  methylene- 
blue: 

Concentrated  alcoholic  solution  of  methylene-blue.  .     3Oc.c. 
Potassium     hydrate      (caustic     potash)      1-10,000 
watery   solution 100  c.c. 

Loffler's  methylene-blue  is  a  good  stain  for  general  pur- 
poses. It  is  perhaps  more  in  use  than  any  other  formula 
for  coloring  the  diphtheria  bacillus. 

Aniline-water  Staining  Solutions. — The  intensity  with 
which  aniline  dyes  operate  may  be  increased  by  adding 
aniline  oil  to  the  solution  : 

Aniline  oil  5  c.c. 

Water   100  c.c. 

Mix,  shake  vigorously,  filter;  the  fluid  after  filtration 
should  be  perfectly  clear;  add— 

Alcohol    10  c.c. 

Alcoholic  solution  of  fuchsin   (or  gentian-violet,  or 
methylene-blue)     1 1  c.c. 

Aniline-water  staining  solutions  do  not  keep  well,  and 
need  to  be  freshly  prepared  about  every  two  weeks.  The 
applications  of  the  aniline-water  stains  will  be  given  under 
separate  headings.  In  general,  however,  they  are  em- 
ployed where  a  stain  of  unusual  power  is  required. 

Gram's  Method. — Cover-glass  preparations,  having  been 
prepared  and  fixed  in  the  usual  manner  (see  page  37),  are 
stained  as  follows : 

(a)  Stain  in  aniline-water  gentian-violet  solution,  from 
two  to  five  minutes.     The  intensity  of  the  stain  may  be  in- 
creased by  warming  slightly. 

(b)  Iodine  solution,  one  and  one-half  minutes  : 


42  MANUAL    OF    BACTERIOLOGY. 

Iodine     I  gram. 

Potassium  iodide   2  grams. 

Water    300  c.c. 

In  this  solution  the  preparation  becomes  nearly  black. 

(c)  Wash   in   alcohol   repeatedly;   the   alcohol   becomes 
stained  with  clouds  of  violet  coloring  matter;  the  alcohol 
is  used  as  long  as  the  violet  color  continues  to  come  away, 
and  until  the  preparation  is  decolorized  or  has  only  a  faint 
steel-blue  color. 

(d)  When  desired,  the  specimens  may  be  stained,  by  way 
of  contrast,  with  a  watery  solution  of  Bismarck  brown  or 
eosin. 

(e)  Wash  in  water,  and  examine  either  in  water  directly 
or  after  drying  and  mounting  in  Canada  balsam.     [A  modi- 
fication of  this  method,  sometimes  called  the  Gram-Gunther 
method,  differs  from  the  preceding  by  using  a  3  per  cent, 
solution  of  hydrochloric  acid  in  alcohol  for  ten  seconds  to 
hasten  decolorization,  washing  in  pure  alcohol  before  and 
after  the  acid  alcohol.     Decolorization  is  more  intense  than 
by  the   Gram   method;   the   diphtheria   bacillus,    which    is 
stained  by  Gram's  method,   is   decolorized  by  the  Gram- 
Gunther  (Kruse).]     The  advantages  of  Gram's  method  are 
that  with  it  certain  bacteria  are  stained  a  violet  color  witli 
more  or  less  intensity  and  other  bacteria  are  not  stained  at 
all.    To  some  extent,  then,  it  furnishes  a  means  of  diagnosis. 

List  of  some  of  the  important  bacteria  that  are  stained 
by  Gram's  method : 

Staphylococcus  pyogenes  aureus. 

Streptococcus  pyogenes, 

Micrococcus  lanceolatus  (of  pneumonia), 

Micrococcus  tetragenus, 

Bacillus  of  diphtheria. 

Bacillus  of  tuberculosis, 

Bacillus  of  leprosy, 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     43 

Bacillus  of  anthrax, 

Bacillus  of  tetanus, 

Bacillus  aerogenes  capsulatus, 

Ray  fungus  of  actinomycosis. 

The  following  bacteria  are  not  stained  by  Gram's  method : 

Gonococcus, 

Diplococcus  intracellularis  meningitidis, 

Micrococcus  melitensis, 

Bacillus  of  chancroid  (Ducrey), 

Bacillus  of  dysentery  (Shiga), 

Bacillus  of  typhoid  fever, 

Bacillus  coli  communis, 

Bacillus  pyocyaneus, 

Bacillus  of  influenza, 

Bacillus  of  bubonic  plague, 

Bacillus  of  glanders   (bacillus  mallei), 

Bacillus  of  malignant  edema, 

Bacillus  of  Friedlander, 

Bacillus  proteus, 

Spirillum  of  Asiatic  cholera, 

Spirillum  of  relapsing  fever. 

Staining  the  Bacillus  of  Tuberculosis. — A  very  large 
number  of  methods  have  been  proposed  for  staining  the 
bacillus  tuberculosis,  all  of  which  depend  upon  the  princi- 
ple that,  after  adding  to  solutions  of  aniline  dyes  certain 
substances,  like  aniline-water,  carbolic  acid,  or  solutions 
of  ammonia  or  soda,  the  bacillus  tuberculosis  is  stained 
with  great  intensity,  and  gives  up  its  stain  with  difficulty. 
Solutions  of  acids  will  remove  the  stain  from  all  parts  of 
the  preparation  excepting  from  the  tubercle  bacilli,  which 
retain  the  dye  having  once  acquired  it.  The  rest  of  the 
preparation  may  now  be  given  a  different  color — contrast- 
stain. 

Bacilli  that  resist  decolorization  by  acids  are  called  acid- 


44  MANUAL    OF    BACTERIOLOGY. 

proof  or  acid-fast.  The  most  important  are  tubercle  and 
leprosy  bacilli.  There  are  various  other  species  however 
most  of  which  are  less  resistant  to  acids  and  alcohol  than 
tubercle  bacilli.  They  are  discussed  in  the  article  on  the 
bacillus  tuberculosis  in  Part  IV. 

Occasionally  spores  of  other  bacteria,  micrococci  and 
horny  epithelial  cells  are  imperfectly  decolorized,  but  their 
forms  distinguish  them  from  tubercle  bacilli.  Minute 
crystalline  needles  which  have  a  shape  like  that  of  bacilli, 
are  often  encountered  in  sputum,  but  their  nature  will  be 
recognized  after  a  little  practice. 

The  stain  for  tubercle  bacilli  is  most  frequently  used  for 
specimens  of  sputum  from  cases  of  suspected  pulmonary 
tuberculosis ;  it  may  be  applied  to  other  fluids  and  secretions 
equally  well.  It  is  not  reliable,  however,  when  applied  to 
milk,  as  the  oil  present  in  milk  interferes  with  its  operation, 
and  milk  and  its  products  quite  often  contain  other  acid- 
proof  bacilli.  The  smegma  of  the  external  genitals  also  fre- 
quently contains  acid-proof  bacilli  that  are  not  tubercle 
bacilli.  On  this  account  all  fluids  and  discharges  from  the 
genito-urinary  tract  need  to  be  examined  with  particular 
care  not  to  confuse  tubercle  bacilli  with  smegma  bacilli. 
(See  smegma  bacilli  in  Chapter  IV.,  Part  II.) 

Patients  should  be  given  minute  instructions  concerning 
the  collection  of  sputum.  The  bottle  used  should  be  new, 
wide-mouthed,  clean,  and  kept  tightly  stoppered  with  a 
clean  cork.  The  patient  should  be  cautioned  against  allow- 
ing the  expectoration  to  get  on  the  outside  of  the  bottle. 
Probably  whatever  risk  is  incurred  by  those  who  examine 
sputum  comes  chiefly  from  the  outside  of  the  bottle  having 
been  soiled  with  sputum  containing  tubercle  bacilli.  Often 
little  white  particles  may  be  seen  floating  in  the  mucous  por- 
tions of  the  sputum.  These  particles  should  be  selected  for 
the  investigation,  and  may  be  spread  in  a  thin  film  on  the 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     45 

cover-glass  with  the  platinum  wire,  which  is  sterilized  in  the 
flame  before  and  after  using.  The  selection  of  the  little 
white  particles  will  be  facilitated  if  the  sputum  be  poured 
into  a  clean  glass  dish,  which  may  be  placed  on  a  black  sur- 
face. A  form  of  porcelain  dish  is  furnished  by  dealers,  the 
bottom  of  which  is  black,  and  which  is  convenient,  for  these 
manipulations.  The  smears  must  be  made  thin,  or  the  sub- 
sequent decolorization,  after  staining,  will  not  be  uniform. 
It  is  hardly  necessary  to  observe  that  the  operator  must  be 
scrupulously  careful  not  to  contaminate  the  material  under 
examination  with  any  kind  of  extraneous  matter.  The 
cover-glasses  and  slides  which  are  used  should  be  new,  and 
should  have  been  cleaned  with  bichromate  of  potassium  and 
sulphuric  acid  (see  page  36). 

When  the  work  is  completed,  the  bottle  containing  the 
sputum  should  be  sterilized  by  steam  or  boiling. 

Many  different  methods  for  staining  the  tubercle  bacillus 
have  been  proposed.  In  most  of  those  now  in  use  the  fol- 
lowing solution  is  employed— 

Fuchsin    i  gram. 

Carbolic  acid,  pure  5  c.c. 

Alcohol     10  c.c. 

Distilled  water  100  c.c. 

The  method  given  below  is  the  one  recommended. 
Method  for  staining  the  tubercle  bacillus: 

(a)  The  cover-glass  preparation  is  made,  dried,  and  fixed 
by  passing  through  the  flame  three  times. 

(b)  The   cover-glass,   held   in    forceps   or   in   a   watch- 
crystal   is   covered   with   steaming  carbol-fuchsin    for   five 
minutes. 

(c)  Wash  in  water. 

(d)  Wash  in  alcohol  containing  3  per  cent,  of  hydro- 
chloric acid  one  minute,  or  longer  if  necessary  to  remove 
the  red  color. 


46  MANUAL    OF    BACTERIOLOGY. 

(e)  Wash  in  water. 

(f)  Stain  with  methylene-blue  solution    (see  page  41) 
thirty  seconds. 

(g)  Wash  in  water. 

(h)  Examine  in  water  directly,  or  after  drying  and 
mounting  in  Canada  balsam.  Tubercle  bacilli  take  a  brilliant 
red  color;  other  bacteria  and  the  nuclei  of  cells  are  stained 
blue. 

Gabbett's  Method. — This  method  is  very  popular  and 
widely  used  on  account  of  its  convenience.  It  is  not  as 
reliable  as  the  one  just  given. 

Gabbett's  solution : 

Methylene-blue    I  to  2  grams. 

25  per  cent,  watery  solution  of  sulphuric  acid.     looc.c. 

(a)  The  cover-glass  preparation  is  to  be  made,  dried,  and 
fixed  by  passing  through  the  flame  three  times. 

(b)  The  carbol-fuchsin  stain  is  applied  from  two  to  five 
minutes  to  the  cover-glass,  held  in  forceps  or  in  a  watch- 
crystal  ;  it  need  not  be  warmed. 

(c)  Wash  in  water. 

(d)  Gabbett's  solution  is  applied  for  one  minute. 

(e)  Wash  in  water.    The  preparation  should  have  a  blue 
color.     It  may  be  examined  in  water  directly  or  after  dry- 
ing and  mounting  in  Canada  balsam. 

Gabbett's  method  has  the  advantage  of  decolorizing  the 
preparation  and  staining  the  background  with  methylene- 
blue  at  the  same  time.  Tubercle  bacilli  are  colored  a 
brilliant  red;  most  other  bacteria  and  the  nuclei  of  cells  are 
colored  blue.  The  acid-proof  bacilli  mentioned  on  page  44 
would  keep  the  red  stain  also,  in  most  cases,  and  would  prob- 
ably be  confused  with  tubercle  bacilli. 

Of  the  numerous  methods  of  staining  tubercle  bacilli  only 
a  few  others  can  be  mentioned.  Aniline-water  fuchsin, 
aniline-water  gentian-violet,  or  carbol-fuchsin  may  be  used. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     47 

The  intensity  of  the  stain  must  then  be  increased  by  warm- 
ing the  preparation  till  it  steams  or  boils,  then  allowing  the 
warm  stain  to  act  on  the  specimens  for  from  three  to  five 
minutes;  the  preparation  may  also  be  left  in  the  cold  stain 
over  night.  Decolorization  may  be  effected  with  a  25  per 
cent,  solution  of  sulphuric  acid  used  till  the  red  color  disap- 
pears, or  a  30  per  cent,  solution  of  nitric  acid,  which  operates 
very  rapidly.  If  the  red  color  persists  after  washing  in 
water,  dip  in  the  acid  again.  After  either  acid  the  prepara- 
tion is  to  be  washed  in  alcohol  until  the'  last  trace  of  the 
stain  has  been  removed.  An  excellent  decolorizing  agent 
is  a  3  per  cent,  solution  of  hydrochloric  acid  in  alcohol, 
used  for  about  a  minute.  With  any  of  these  acid  solutions 
the  decolorization  can  be  accomplished  more  perfectly  than 
with  Gabbett's  solution,  where  the  operation  of  the  de- 
colorizing agent  is  masked.  The  contrast-stain  may  be 
omitted  entirely  if  it  is  desired.  A  suitable  contrast-stain 
after  fuchsin  staining  is  a  solution  of  methylene-blue ;  after 
gentian-violet  staining,  Bismarck  brown. 

Those  who  have  had  experience  in  staining  tubercle  ba- 
cilli soon  discover  that  the  bacilli  exhibit  some  differences 
in  their  resisting  power  to  strong  acids.  One  encounters 
occasionally  bacilli  that  are  perfectly  stained  side  by  side 
with  others  that  are  more  or  less  completely  decolorized. 
These  facts  show  the  necessity  of  practice  with  any  method, 
and  of  exercising  caution  and  judgment  in  making  a' diag- 
nosis where  the  number  of  bacilli  happens  to  be  scanty. 
If  tubercle  bacilli  are  not  found  in  the  first  preparation, 
other  preparations  should  be  made.  Sometimes  a  large 
number  of  cover-glasses  must  be  examined. 

Various  expedients  have  been  devised  to  concentrate 
tubercle  bacilli  when  only  a  small  number  may  be  present 
in  a  sample  of  sputum.  In  Biedert's  method  about  15  c.c. 
of  sputum  are  mixed  with  5  c.c.  of  distilled  water,  4  to  8 


48  MANUAL    OF    BACTERIOLOGY. 

drops  of  sodium  hydrate  solution  are  added,  and  the  mix- 
ture is  boiled.  After  boiling,  add  about  15  c.c.  of  distilled 
water.  The  mixture  may  be  set  aside  in  a  conical  glass 
for  from  twenty-four  to  forty-eight  hours  when  the  sedi- 
ment may  be  collected,  smeared  on  a  cover-glass  and 
stained  for  tubercle  bacilli ;  or  the  sediment  may  be  precip- 
itated rapidly  by  the  use  of  the  centrifuge.  The  sediment 
will  be  found  to  have  little  adhesive  power,  and  will  not 
stick  well  to  the  cover-glass.  It  is  convenient  to  save  some 
of  the  original  sputum  and  mix  it  with  the  sediment  for 
this  purpose. 

Staining  Bacteria  in  Tissues. — Pieces  of  organs  about 
i  cm.  in  thickness  may  be  taken.  Alcohol  is  the  best 
agent  for  preserving  them.  The  hardening  will  be  com- 
pleted in  a  few  days.  It  is  best  to  change  the  alcohol. 
The  amount  of  the  alcohol  must  be  twenty  times  the  bulk 
of  the  tissue  to  be  preserved. 

Ten  parts  of  the  standard  40  per  cent,  solution  of  form- 
aldehyde, with  90  parts  water  make  a  good  mixture  for 
fixation ;  after  twenty-four  hours  change  to  alcohol. 

Imbedding  in  Collodion  or  Celloidin. — From  alcohol  the 
pieces  of  tissue  are  placed  in  equal  parts  of  alcohol  and 
ether  twenty-four  hours;  thin  collodion  (i^  per  cent.), 
twenty-four  hours ;  thick  collodion  of  a  syrupy  consistency 
(6  per  cent.)  twenty- four  hours.  The  specimen  is  laid  upon 
a  block  of  wood  and  surrounded  by  thick  collodion,  and  then 
inverted  in  70  per  cent,  alcohol.  The  collodion  makes 
a  firm  mass,  surrounding  and  permeating  the  tissue,  and 
permits  very  thin  sections  to  be  cut.  The  soluble  cotton 
sold  by  dealers  in  photographer's  supplies  serves  as  well  as 
the  expensive  preparation  known  as  celloidin.  To  make 
collodion,  dissolve  it  in  equal  parts  of  alcohol  and  ether. 
Soluble  cotton  is  also  called  pyroxylin,  and  is  a  kind  of 
gun-cotton. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     49 

Imbedding  in  Paraffin.  —  (a)  Pieces  of  tissue  2  to  3  mm. 
thick  which  have  already  been  fixed  in  alcohol  or  formal- 
dehyde are  to  be  placed  in  absolute  alcohol  for  twenty-four 
hours. 

(b)  In  pure  xylol  one  to  three  hours. 

(c)  In  a  saturated  solution  of  paraffin  in  xylol  one  to 
three  hours. 

(d)  In  melted  paraffin  having  a  melting-point  of  50°  C, 
which  requires  the  use  of  a  water-bath  or  oven,  one  to  three 
hours.     The  xylol  must  be  entirely  driven  off,  and  the  tis- 
sue thoroughly  infiltrated. 

(e)  Change  to  fresh  paraffin  for  one  hour. 

(/)  Finally,  place  the  tissue  in  a  small  dish  or  paper 
box  and  pour  the  melted  paraffin  about  it.  Harden  as 
quickly  as  possible  with  running  water.  It  is  important  to 
fix  the  piece  of  tissue  in  a  suitable  position,  if  the  position 
is  of  importance,  before  pouring  in  the  melted  paraffin. 

Sections  of  exquisite  thinness  may  now  be  cut.  The 
knife  need  not  be  wet.  Paraffin  imbedding  is  especially 
desirable  when  serial  sections  are  to  be  made. 

In  order  to  mount  the  sections,  proceed  as  follows : 

(a)  Place  the  sections  on  water  in  a  porcelain  capsule. 
Warm  slightly,  when  the  sections  will  flatten  nicely.    Smear 
the  surface  of  a  slide  with  a  very  thin  layer  of  Mayer's 
glycerin-albumen  mixture.      Dip  the  slide  under  the   sec- 
tions; lift  them;  and  then  drain  off  the  water,  leaving  the 
sections  in  their  proper  positions.     Let  them  dry  for  some 
hours  in  the  incubator,  and  they  will  be  firmly  fastened  to 
the  slide. 

(b)  Dissolve  out  the  paraffin  in  one  of  the  numerous  sol- 
vents (xylol,  a  few  minutes). 

(c)  At  this  point  the  xylol  should  be  washed  off  with 
absolute  alcohol,  and 

(d)  The  section  is  stained. 


50  MANUAL    OF    BACTERIOLOGY. 

(e)   Dehydrate  in  absolute  alcohol. 
(/)   Clear  in  xylol. 
(g)   Mount  in  balsam. 

GLYCERIN-ALBUMEN   MIXTURE    (MAYER). 

Equal  parts  of  white  of  egg  and  glycerin  are  thoroughly  mixed,  and 
then  filtered.  Add  a  little  gum-camphor  to  preserve. 

Section  Cutting. — Cutting  is  best  done  with  an  instru- 
ment called  a  microtome.  The  tissues  may  be  imbedded  in 
collodion  or  paraffin ;  or  when  they  have  been  hardened  with 
formaldehyde  they  may  be  cut  after  freezing.  Bacteria 

Fie.    10. 


Schanze  microtome. 

stain  admirably  in  fro/en  sections.  Kor  routine  work  col- 
lodion imbedding  will  be  found  as  convenient  a  process  as 
any.  Paraffin  imbedding  gives  the  thinnest  sections. 

A  microtome  consists  of  a  heavy,  sliding  knife-carrier, 
which  moves  with  great  precision  on  a  level,  and  of  a  de- 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     5! 

vice  for  elevating  the  object  which  is  to  be  cut  any  desired 
distance  after  each  excursion  of  the  knife.  The  thickness 
of  the  section  will  be  the  distance  which  the  object  is  ele- 
vated. The  knife  is  kept  wet  with  alcohol  during  the  cut- 
ting of  collodion  sections,  otherwise  it  is  left  dry.  The 
microtome  is  usually  provided  with  a  special  form  of  knife. 
A  razor  will  serve  nearly  as  well,  after  having  had  the 
lower  side  ground  flat.  If  a  razor  is  used,  a  special  form 
of  razor-holder  must  be  attached  to  the  microtome  to  re- 
ceive the  razor.  Above  all,  it  is  necessary  that  the  knives 
should  be  kept  in  good  condition.  Only  occasionally  will 
they  need  honing,  using  a  fine  water-stone  or  Belgian 
hone.  The  movement  in  honing  should  be  from  heel  to 
toe,  always  placing  the  back  of  the  knife  next  the  hone 
when  turning.  The  knife  should  be  stropped  frequently. 
The  leather  of  the  strop  should  be  glued  to  a  strip  of  wood 
to  make  a  flat  surface.  The  movement  in  stropping  should 
be  from  toe  to  heel.  Sections  should  be  cut  to  a  thickness 
of  not  more  than  25^.  Thinner  sections  (5  to  io/*)  are  to 
be  desired. 

Staining  of  Sections. — A  watery  solution  of  one  of  the 
aniline  dyes  is  used — fuchsin,  gentian-violet  or  methylene- 
blue — made  by  adding  a  few  drops  of  the  alcoholic  solution 
to  a  dish  filled  with  water.  Loffler's  solution  of  methylene- 
blue  serves  very  well. 

By  this  process  most  bacteria  are  stained ;  also  the  nuclei 
of  cells;  frequently,  also,  certain  granules  contained  within 
some  cells  (German,  Mastzcllcn},  which  may  easily  be 
mistaken  for  bacteria  by  the  inexperienced  (basophilic 
granules). 

(a)  Place  the  section  in  the  staining  solution  from  two  to 
five  minutes. 

(b)  Wash  in  water. 
5 


52  MANUAL    OF    BACTERIOLOGY. 

(c)  Place  in  a  watery  solution  of  acetic  acid,  .1  per  cent., 
for  one  minute. 

(d)  Alcohol,  one  to  two  minutes;  change  to  absolute  al- 
cohol.    Touch  the  sections  to  blotting-paper  to  remove  the 
superfluous  alcohol. 

(e)  Xylol  until  clear;  xylol  is  to  be  preferred  to  other 
clearing  agents,  like  oil  of  cloves,  most  of  which  slowly  re- 
move aniline  colors.    It  has  the  disadvantage  of  not  clearing 
when  the  slightest  trace  of  water  is  present ;  dehydration  in 
alcohol  must,  therefore,  be  complete.     The  section  should 
be  removed  from  the  xylol  as  soon  as  it  is  cleared ;  otherwise 
wrinkling  occurs. 

(f)  The  section  is  placed  upon  a  glass  slide;  a  drop  of 
Canada  balsam  is  placed  upon  it  and  then  a  cover-glass. 
The  Canada  balsam  should  be  dissolved  in  xylol. 

The  section  is  to  be  manipulated  with  straight  or  bent 
needles.  The  removal  from  xylol  to  the  glass  slide  is  man- 
aged best  with  a  spatula  or  section-lifter. 

The  above  statements  apply  to  frozen  sections  or  to  sec- 
tions imbedded  in  celloidin.  Paraffin  sections  are  preferably 
attached  to  the  slide  with  glycerin-albumen.  The  different 
steps  in  the  process  follow  in  the  same  order.  The  stain 
may  be  poured  on  the  slide,  or  the  slide  may  be  placed  in 
a  large  dish  full  of  staining  fluid.  (See  page  49.)  Celloidin 
sections  may  also  be  stained  on  the  slide.  If  the  section  be 
well  spread  and  flattened  thoroughly  with  blotting-paper, 
it  will  usually  adhere  to  the  slide,  and  is  less  likely  to 
wrinkle.  It  must  not  be  allowed  to  dry. 

Gram's  Method  may  be  applied  to  the  staining  of  sec- 
tions of  tissues  as  well  as  to  smears  upon  cover-glasses. 

(a)  Place  the  section  in  aniline-water  gentian-violet, 
one  to  five  minutes. 

(&)    Rinse  briefly  in  water. 

(c)  Iodine  solution  (see  page  42),  one  and  one-half 
minutes. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     53 

(d)  Alcohol,  until  decolorized  to  a  faint  blue-gray. 

(e)  Xylol. 

(f)  Mount  on  a  slide  in  balsam. 

Weigert's  Modification  of  Gram's  Method,  or  Weigert's 
Stain  for  Fibrin.  —  (a)  Place  the  section  in  aniline-water 
gentian-violet  solution,  five  minutes  or  more. 

(&)   Wash  briefly  in  water. 

(c)  Place  the  section  upon  a  slide  by  means  of  a  section- 
lifter;  having  straightened  it  carefully,  absorb  the  water 
with  blotting-paper. 

(d)  Iodine  solution  (see  page  42)  one  to  two  minutes. 

(e)  Absorb  the  iodine  solution  with  blotting-paper. 

(f)  Add  aniline  oil,  removing  it  from  time  to  time  with 
blotting-paper,  and  adding  fresh  aniline  oil  until  the  color 
ceases  to  come  away.     (Aniline  oil  serves  in  this  connection 
both  to  decolorize  and  to  dehydrate.     It  absorbs  the  water 
rapidly  and  efficiently.     However,  on  account  of  its  decol- 
orizing tendency,  it  must  be  removed  before  the  specimens 
can  be  mounted  permanently.) 

(g)  Add  xylol;  remove  it  with  blotting-paper;  and  add 
fresh  xylol  several  times,  in  order  to  extract  the  last  trace 
of  aniline  oil. 

(h)   Mount  in  Canada  balsam. 

This  method  is  more  convenient  for  the  staining  of  sec- 
tions than  the  Gram  method.  The  results,  however,  are 
essentially  the  same  as  far  as  the  bacteria  are  concerned ; 
fibrin  and  hyaline  material  are  stained  blue,  bacteria  violet. 
It  is  often  impossible  to  decolorize  the  nuclei  completely 
without  decolorizing  the  bacteria  also.  The  parts  of  the 
nuclei  which  remain  stained  often  present  pictures  that  re- 
semble bacteria,  and  which  may  lead  to  error  if  not  recog- 
nized. Basophilic  granules  also  retain  the  stain,  as  do  the 
horny  cells  of  the  epidermis.  These  remarks  apply  also 
to  Gram's  method,  except  as  regards  fibrin.  Very  beauti- 


54  MANUAL    OF    BACTERIOLOGY. 

ful  preparations  can  be  obtained  according  to  this  or  the 
Gram  method  when  the  sections  have  previously  been 
stained  in  carmine;  the  nuclei  will  then  be  colored  red, 
bacteria  violet. 

Tubercle  bacilli  may  be  stained  in  sections  as  follows : 

(a)  Use  carbol-fuchsin,   or  aniline-water   gentian-violet 
for  one-half  to  two  hours  with  very  gentle  warming,  or 
over  night  without  warming. 

(b)  Wash  in  water. 

(c)  Decolorize  with  some  one  of  the  decolorizing  agents 
mentioned  in  connection  with  the  staining  of  tubercle  ba- 
cilli in  cover-glass  preparations,  preferably  3  per  cent,  hy- 
drochloric acid  alcohol.     Decolorization  must  be  continued 
until  the  red  color  has  disappeared,  which  requires  one-half 
to  several  minutes. 

(d)  Wash  in  alcohol. 

(e)  Wash  in  water. 

(/)    Use    hematoxylin    as    a    contrast-stain    for    fuchsin 
preparations,  and  carmine  for  gentian-violet  preparations. 
(It  is  better  to  stain  with  carmine  first  of  all  and  before 
staining  the  bacilli.     The  carmine  is  not  affected  by  the 
subsequent  treatment. ) 
(g)   Wash  in  water. 
O)   Alcohol, 
(i)   Xylol. 
(/)   Balsam. 

Nuclear  stains,  which  may  be  used  as  contrast-stains  for 
sections : 

DELAFIELD'S  HEMATOXYLIX. 

Hematoxylin    crystals 4  grams. 

Alcohol     25  c.c. 

Ammonia  alum    50  grams. 

Water    400  c.c. 

Glycerin    TOO  c.c. 

Methyl  alcohol   100  c.c. 


EXAMINATION  OF  BACTERIA  WITH  THE  MICROSCOPE.     55 

Dissolve  the  hematoxylin  in  the  alcohol,  and  the  am- 
monia alum  in  the  water.  Mix  the  two  solutions.  Let 
the  mixture  stand  four  or  five  days  uncovered;  it  should 
have  become  a  deep  purple.  Filter  and  add  the  glycerin 
and  the  methyl  alcohol.  After  it  has  become  dark  enough, 
filter  again.  Keep  it  a  month  or  longer  before  using;  the 
solution  improves  with  age.  At  the  time  of  using,  filter 
and  dilute  with  water  as  desired. 

LITHIUM-CARMINE    (ORTH). 

Carmine    2.5  grams. 

Saturated  watery  solution  of  lithium  carbonate.    loo.oc.c. 

Add  a  few  crystals  of  thymol.  The  carmine  dissolves 
readily  in  the  lithium  carbonate  solution.  Filter  the  stain 
at  the  time  of  using.  Sections  are  to  be  left  in  the  stain 
five  to  twenty  minutes. 

Sections  stained  in  carmine  are  placed  directly  in  acid 
alcohol  (i  part  hydrochloric  acid,  100  parts  70  per  cent. 
alcohol)  for  five  to  ten  minutes.  They  acquire  a  brilliant 
scarlet  color.  When  used  as  a  contrast-stain  for  tissues 
containing  bacteria,  it  is  best  to  use  it  before  staining  the 
bacteria,  which  might  be  decolorized  by  the  acid  alcohol. 

Staining  of  Blood-Films. — The  method  of  Wright  is 
the  one  recommended.  It  is  applicable  to  bacteria  and  to  the 
parasite  of  malaria,  and  is  useful  as  a  general  stain  for 
blood.  Films  of  blood  are  prepared  as  directed  in  chapter 
VII.,  Part  I.,  and  are  allowed  to  dry. 

(a)  The  stain  is  poured  over  the  surface  of  the  prepara- 
tion till  it  covers  it.  This  serves  to  fix  the  film  of  blood.  It 
is  allowed  to  remain  for  one  minute. 

(6)  Add  distilled  water,  drop  by  drop,  till  a  reddish  tint 
appears  at  the  edges  and  a  metallic  scum  forms  on  the  sur- 
face. About  six  drops  are  needed  for  a  three-fourths  inch 
cover-glass.  The  real  staining  of  the  preparation  now  takes 
place,  and  requires  two  or  three  minutes. 


56  MANUAL    OF    BACTERIOLOGY. 

(c)  Wash  in  distilled  water  till  the  thin  parts  of  the 
preparation  have  a  yellowish  or  pinkish  tint,  which  requires 
one  to  three  minutes. 

(d)  Dry  with  blotting-paper  and  mount  in  Canada  bal- 
sam. 

Bacteria,  malarial  parasites,  and  cell-nuclei  are  stained 
blue,  red  blood-corpuscles  are  orange-pink,  while  the  specific 
granules  of  the  leucocytes  (neutrophilic,  etc.)  appear  in 
various  tints  from  red  to  dark  blue.  The  chromatin  of  the 
malarial  parasite  takes  a  lilac  to  red  color.  The  blood-plates 
have  a  bluish  or  purplish  color  and  must  not  be  confused 
with  malarial  parasites. 

The  staining  fluid  is  prepared  as  follows :  To  100  c.c.  of  a  one  per 
cent,  solution  of  sodium  bicarbonate  in  water  add  i  gram  of  methyl  ene- 
blue.  Place  in  the  steam  sterilizer  at  ioo°C.  for  one  hour.  When  cool 
add  one-tenth  per  cent,  watery  solution  of  eosin  (Griibler,  yellowish, 
soluble  in  water)  until  the  mixture  loses  its  blue  color,  becomes  pur- 
ple, and  a  metallic  scum  forms  on  the  surface.  About  500  c.c.  of  the 
eosin  solution  are  needed.  Collect  the  precipitate  on  a  filter ;  let  it  dry ; 
make  a  saturated  solution  of  the  precipitate  in  methyl  alcohol;  filter. 
To  the  quantity  obtained  add  one-fourth  as  much  methyl  alcohol,  so 
that  the  solution  may  not  be  completely  saturated.  The  purpose  of  the 
above  procedures  is  to  modify  the  methylene-bluc  so  that  other  stain- 
ing elements  are  developed  in  it  (polychromism).  The  modified 
methylene-blue  solution  is  then  combined  with  eosin.  For  full  details 
see  Wright,  Journal  of  Medical  Research,  Vol.  VII.  1902. 

Staining  of  Spores. — The  method  is  applicable  to  cover- 
glass  preparations  which  may  be  prepared  in  the  usual 
way  from  material  supposed  to  contain  spores. 

(a)  After  drying  the  smear  on  the  cover-glass,  and  fixa- 
tion with  heat  by  passing  through  the  flame  three  times, 
use  as  a  stain  aniline-water  fuchsin. 

(b)  Heat  until  the  preparation  begins  to  boil;  remove 
for  a  minute;  heat  again,  and  again  remove;  repeat  this 
process  six  times. 

(c)  Wash  in  3  per  cent,  hydrochloric  acid  alcohol   one 
minute,  or  less. 


f  ' 

EXAMINATION  OF    BACTERIA  WITH  THE  MICROSCOPE.    57 

(d)  Wash  in  water. 

(e)  Stain  with  watery  solution  of  methylene-blue  half  a 
minute. 

(/)   Wash. 

(£)    Dry. 

(h)   Balsam. 

The  spores  are  intensely  stained  by  the  fuchsin.  The 
stain  is  removed  from  everything  except  the  spores  by  the 
acid  alcohol.  The  methylene-blue  solution  stains  the  bodies 
of  the  bacteria,  the  spores  remaining  brilliant  red.  There 
are  various  other  methods  for  staining  spores,  but  this  pro- 
cedure gives  good  results.  The  principle  is  the  same  as  in 
staining  the  tubercle  bacillus,  except  that  more  pains  are 
needed  to  impregnate  spores  with  the  dye. 

Staining  of  Capsules. — The  capsules  which  many  bac- 
teria possess,  appear  to  be  made  of  some  gelatinous  sub- 
stance, which  is  difficult  to  stain. 

Method  of  Welch. —  (a)  Cover-glass  preparations  are 
made  in  the  usual  manner.  Pour  glacial  acetic  acid  over 
the  film. 

(&)  After  a  few  seconds,  replace  with  anilin- water  gen- 
tian-violet, without  washing  in  water.  Change  the  stain 
several  times  to  remove  all  the  acetic  acid.  Allow  it  to  act 
three  or  four  minutes. 

(c)  Wash  and  examine  in  salt  solution,  0.8  to  2.0  per 
cent. 

Bacteria  are  deeply  stained,  while  their  capsules  are  pale 
violet.  This  method  has  been  recommended  for  staining 
the  capsule  of  the  pneumococcus. 

Methods  of  Hiss. — i.  (a)  Cover-glass  preparations  are 
made  in  the  usual  manner,  and  fixed  in  the  flame. 

(&)  Stain  for  a  few  seconds  in  a  half-saturated  watery 
solution  of  gentian-violet. 

(c)  Wash  in  25  per  cent,  solution  of  potassium  carbonate 
in  water. 


58  MANUAL    OF    BACTERIOLOGY. 

(d)   Mount  and  study  in  the  same. 

2.  (a)  Cover-glass  preparations  are  made  and  fixed  in 
the  ordinary  way. 

(b)  Use  the  following  stain,  heated  till  it  steams: 

Saturated  alcoholic  solution  of  gentian-violet  or  fuchsin.     5  c.c. 
Distilled  water  95  c.c. 

(c)  Wash  in   20  per  cent,   solution  of  cupric  sulphate 
crystals. 

(d)  Dry  and  mount  in  Canada  balsam. 

The  methods  of  Hiss  are  recommended  to  be  used  for 
bacteria  that  have  been  cultivated  on  serum-agar  with  i  per 
cent.  of  dextrose.  They  have  shown  that  many  streptococci 
have  capsules.  The  writer  has  had  good  success  from  the 
latter  method,  with  preparations  of  the  pneumococcus  from 
animal  tissues. 

Staining  of  Flagella. — Flagella  are  among  the  most 
difficult  of  all  objects  to  stain.  The  best-known  method  is 
that  of  Lofllcr.  It  is  important  to  use  young  cultures,  pref- 
erably on  agar. 

(a)  A  small  portion  of  the  culture  is  mixed  on  a  cover- 
glass  with  a  drop  of  water.     The  preparations  must  be  ex- 
ceedingly thin.     The  mixing  must  be  done  with  care   in 
order  not  to  break  off  the  delicate  flagella.     The  cover- 
glass  must  be  perfectly  clean,  see  page  36. 

(b)  After  drying,  fixation  is  effected  by  passing  through 
the  flame  three  times. 

(c)  The  essential  point  in  this  method  is  the  use  of  a 
mordant  as  follows : 

Tannic  acid,  20  per  cent,  solution 10  c.c. 

Saturated  solution  of  ferrous  sulphate 5  c-c- 

Saturated  alcoholic  solution  of  fuchsin i  c.c. 

This  solution  is  filtered  and  a  few  drops  are  placed  on 
the  cover-glass,  or  the  cover-glass  is  placed,  face  down,  in 


EXAMINATION   OF    BACTERIA  WITH   THE   MICROSCOPE.    59 

a  dish  containing  the  stain ;  it  is  then  left  tor  one  to  five  min- 
utes, warming  slightly. 

(d)  Wash  in  water. 

(e)  Stain  with  aniline-water  fuchsin,  or  carbol-fuchsin. 
(/)   Wash  in  water. 

(£)   Dry. 

(h)   Mount  in  Canada  balsam. 

(According  to  Loffler,  certain  bacteria  require  the  addi- 
tion of  an  acid  solution,  and  certain  others  an  alkaline 
solution,  but  many  observers  consider  this  unnecessary.) 

Another  and  very  valuable  method  is  that  of  Van  Er- 
mengem. 

(a)  Make  and  fix  cover-glass  preparations  as  in  the  pre- 
ceding method. 

(b)  Use  the   following  mordant   for  one-half  hour  at 
room-temperature  or  for  five  minutes  at  50°  to  60°  C. 

Osmic  acid  2  per  cent,   solution I 

Tannic  acid  10  to  25  per  cent,  solution 2 

(c)  Wash  carefully  in  distilled  water  and  then  in  alcohol. 

(d)  Place  for  a  few  seconds  in  a  0.25  to  0.50  per  cent, 
solution  of  nitrate  of  silver — "  the  sensitizing  bath." 

(e)  Without  washing  transfer  to  the  "  reducing  and  re- 
inforcing bath  " : 

Gallic   acid    5  grams. 

Tannic  acid    3  grams. 

Fused  potassium  acetate   10  grams. 

Distilled    water    350  c.c. 

(/)  After  a  few  seconds,  replace  the  preparation  in  the 
nitrate  of  silver  solution,  in  which  it  is  kept  constantly 
moving,  till  the  solution  begins  to  acquire  a  brown  or  black 
color. 

Some  recommend  leaving  the  preparation  in  the  nitrate 
of  silver  solution  for  two  minutes  in  the  first  place,  and  in 
6 


60  MANUAL    OF    BACTERIOLOGY. 

the  reducing  bath  for  two  minutes,  without  using  the  nitrate 
of  silver  solution  a  second  time. 

(g)  Finally  wash  in  distilled  water,  dry,  mount  in 
Canada  balsam.  It  is  difficult  to  avoid  the  formation  of 
precipitates;  otherwise  the  results  of  this  method  are  usu- 
ally good. 


("  * 

STERILIZATION.  6 1 


CHAPTER   II. 

STERILIZATION. 

BY  sterilization  is  meant  the  killing  of  all  microorgan- 
isms found  on  or  in  any  body  or  substance.  It  is  possible 
to  sterilize  objects  by  the  use  of  bichloride  of  mercury  (cor- 
rosive sublimate),  carbolic  acid  and  other  chemical  agents. 
Sterilization  is  usually  accomplished  by  heat.  The  most 
effective  sterilization  is  that  done  by  steam  and  by  borling; 
they  are  not,  however,  suitable  for  all  kinds  of  material. 

The  naked  flame  of  the  Bunsen  burner  or  the  alcohol 
lamp  is  used  largely  for  the  sterilization  of  small  articles. 
It  is  evident  that  no  more  efficient  way  of  sterilization  could 
be  devised  than  by  burning  objects,  or  subjecting  them  to 
a  red  heat.  The  uses  of  this  method  will  at  once  suggest 
themselves;  for  instance,  surgical  dressings  that  have  be- 
come soiled  with  discharges  and  similar  materials  can  be 
most  easily  disposed  of  by  simply  burning  them  up.  In 
laboratory  work  the  flame  is  constantly  employed  for  the 
sterilization  of  the  platinum  wire,  forceps,  pipettes  and 
cover-glasses;  occasionally  test-tubes  are  sterilized  in  this 
manner. 

Hot-Air  Sterilization. — Hot  air,  at  a  temperature  of  150° 
C,  or  higher,  maintained  for  an  hour,  is  very  valuable  for 
some  materials  although  less  effective  than  steam.  It  has 
been  found  that  the  spores  of  certain  bacteria  are  not  killed 
even  by  exposure  to  this  temperature,  but  it  is  sufficient  for 
ordinary  conditions.  Hot-air  sterilization  is  employed  for 
glassware  such  as  Petri  dishes,  flasks  and  test-tubes. 
Flasks  and  test-tubes  are  generally  plugged  with  raw  cot- 


62 


MANUAL    OF    BACTERIOLOGY. 


ton.  The  sterilization  should  change  the  cotton  to  a  light 
brown  color,  but  it  should  not  be  scorched  to  a  dark  brown. 
Glassware  should  be  placed  within  the  sterilizer  when  it  is 
cold,  and  after  heating  should  be  allowed  to  cool  gradually 
in  order  to  avoid  breaking.  Hot-air  sterilization  is  never 
used  for  culture-media. 

The  hot-air  sterilizer  is  a  box  made  of  sheet-iron,  the 
walls  being  double,  with  an  air-space  between  them.     On 

FIG.  ii. 


Hot-air  sterilizer. 

one  side  is  a  door.  There  are  openings  at  the  top  to  secure 
the  circulation  of  air  in  the  air-chamber.  A  thermometer 
passes  from  the  top  into  the  interior  of  the  sterilizer  so 
that  one  may  read  off  the  temperature  that  is  being  attained. 
The  sterilizer  should  be  placed  so  that  there  will  be  no  dan- 
ger of  its  setting  fire  to  inflammable  articles,  as  the  heat 


STERILIZATION.  63 

may  occasionally  become  very  intense.  It  is  well,  if  pos- 
sible, to  have  it  fastened  to  a  brick  wall. 

Boiling. — Boiling  is  an  efficient  method  of  sterilization. 
It  is  often  used  for  instruments.  In  laboratory  work  steam 
is  generally  substituted  for  it. 

Steam  Sterilization. — Steam  sterilization  is  the  most 
generally  used  of  all  forms  of  sterilization  and  is  the  most 
effective.  It  is  employed  for  perishable  bodies  which  would 
be  injured  by  dry  air  sterilization  or  by  chemical  ger- 
micides ;  for  example,  it  is  used  for  surgical  instruments  and 
for  culture-media;  in  laboratory  work,  especially  for  cul- 
ture-media. It  has  been  found  that  there  are  some  forms 
of  bacteria  which,  in  the  resting  or  spore  stage,  can  resist 
even  the  action  of  steam  for  several  hours.  Such  pro- 
longed exposure  to  steam  would  be  very  injurious  to  cul- 
ture-media, which  are  more  or  less  unstable  organic  sub- 
stances. What  is  called  fractional,  intermittent  or  dis- 
continuous sterilization  is  used  for  such  materials.  By  that 
plan  the  medium  is  sterilized  with  steam  for  fifteen  minutes 
on  each  of  three  consecutive  days.  The  object  of  inter- 
mittent sterilization  as  explained  by  Tyndall,  who  proposed 
it,  is  this :  The  culture-medium  may  be  supposed  to  contain 
fully  developed  bacteria,  and  also  bacteria  in  the  spore  or 
resting  stage.  The  first  sterilization  of  fifteen  minutes  will 
probably  be  sufficient  to  destroy  all  the  fully  developed  bac- 
teria; during  the  twenty-four  hours  between  the  first  and 
second  sterilization  all  of  the  spores  which  have  survived 
the  first  sterilization  may  be  expected  to  have  become  fully 
developed  into  bacteria  which  can  be  destroyed  by  the 
second  sterilization ;  the  third  sterilization  is  directed  against 
any  spore  forms  which  may  possibly  have  survived  the  sec- 
ond sterilization. 

Although  the  spore  forms  which  are  so  extremely  resist- 
ant are  mostly  non-pathogenic,  as  for  example  the  bacilli  of 


64 


MANUAL   OF    BACTERIOLOGY. 


hay  and  potato,  they  nevertheless  are  capable  of  ruining 
the  culture-media  with  which  one  works. 

It  has  been  shown  by  T.  Smith  that  the  discontinuous 
method  cannot  be  relied  upon  to  sterilize  fluids  in  shallow 
layers  that  are  freely  exposed  to  the  air.  For  if  the  spores 
of  anaerobic  bacteria  happen  to  be  present  in  such  fluids, 
they  will  not  develop  into  the  adult  form  between  the  appli- 
cations of  heat,  under  aerobic  conditions. 

The  form  of  sterilizer  most  widely  used  in  the  United 
States  is  that  which  is  known  as  the  Arnold  Steam  Sterilizer. 

FlC.     12. 


Diagram  of  the  Arnold  steam  sterilizer. 

The  Arnold  sterilizer  consists  of  a  cylinder  of  tin  or 
copper  with  a  cover,  which  is  enclosed  in  a  movable,  cylin- 
drical outer  cover  or  hood.  The  inner  cylinder  has  an 
opening  in  the  bottom  through  which  steam  may  enter,  the 


STERILIZATION.  65 

steam  coming  from  a  small  chamber  underneath  with  a 
copper  bottom  to  which  the  flame  is  applied.  The  peculi- 
arity of  this  form  of  sterilizer  consists  in  the  fact  that  the 
steam  which  escapes  from  the  sterilizing  chamber. will  be 
condensed  beneath  the  outer  cover  or  hood  and  will  fall 
back  upon  the  pan  over  the  chamber  in  which  the  steam  is 
generated.  The  bottom  of  this  pan  is  perforated  with  three 

FIG.   13. 


Steam  sterilizer,  Massachusetts  Board  of  Health. 

small  holes  which  allow  the  water  of  condensation  to  return 
into  the  chamber  where  the  steam  is  generated.  The  ster- 
ilizer will,  therefore,  to  a  certain  extent,  supply  itself  with 
water,  although  not  by  any  means  perfectly.  It  is,  how- 
ever, less  likely  to  boil  dry  than  other  forms  of  sterilizers, 
and  it  has  the  advantage  of  being  reasonably  cheap  and 


66 


MANUAL    OF    BACTERIOLOGY. 


quite  effective.  The  space  enclosed  by  the  hood  also  serves 
as  a  steam-jacket  and  helps  to  overcome  fluctuations  in  tem- 
perature. A  great  improvement  upon  the  ordinary  Arnold 
sterilizer  is  the  modification  of  it  devised  by  the  Massachu- 
setts Board  of  Health. 

In  the  use  of  this,  or  any  form  of  steam  sterilizer,  the 
time  when  sterilization  is  supposed  to  begin  must  be  counted 

FIG.   14. 


Koch's   Steam   Sterilizer. 


as  that  when  boiling  is  brisk  and  it  is  evident  that  the  ster- 
ilizing chamber  is  filled  with  hot  steam;  or,  what  is  better, 
when  the  thermometer  registers  100°  C,  if  the  sterilizer  be 
provided  with  a  thermometer.  With  a  large  Arnold  ster- 
ilizer a  temperature  of  100°  C.  may  not  be  reached  until  it 


STERILIZATION.  67 

has  been  heated  with  a  rose-burner  for  twenty  to  thirty-five 
minutes. 

The  sterilizer  invented  by  Koch  is  still  largely  in  use. 
It  is  a  tall  cylindrical  tin  vessel  covered  with  asbestos  or 
felt.  The  lower  portion  is  filled  with  water;  on  the  side 
is  a  water-gauge  indicating  the  height  of  the  water,  in  order 
that  one  may  observe  when  there  is  danger  of  the  sterilizer 
boiling  dry.  Over  the  top  there  is  a  tight-fitting  cover. 
The  steam  is  generated  by  a  Bunsen  burner  standing  under- 
neath. A  perforated  shelf  placed  some  distance  above  the 
surface  of  the  water  is  for  the  reception  of  the  tubes  and 
flasks  that  are  to  be  sterilized. 

The  sterilization  of  blood-serum  sometimes  has  to  be  per- 
formed in  a  specially  devised  sterilizer,  when  a  clear,  fluid 
medium  is  desired.  In  this  case  the  serum  is  heated  for  an 
hour  on  each  of  six  consecutive  days  to  a  temperature  of 
only  58°  C.  To  obtain  a  transparent  but  solid  medium  the 
serum  is  kept  at  a  temperature  of  75°  C.  for  an  hour  on 
each  of  four  consecutive  days.  The  process  must  be  con- 
ducted carefully  to  avoid  clouding  of  the  serum. 

Pasteurization. — The  name  pasteurization  has  been  ap- 
plied to  the  partial  sterilization  of  substances  at  a  compara- 
tively low  temperature.  It  is  employed  particularly  for 
milk.  The  temperature  used  (70°  to  75°  C.  for  20  to  30 
minutes)  is  sufficient  to  destroy  all  ordinary  pathogenic 
bacteria ;  for  example,  the  bacilli  of  tuberculosis  and  typhoid 
fever.  Furthermore,  the  great  majority  of  the  saprophytic 
bacteria  are  destroyed,  and  milk  which  has  been  pasteurized 
will  remain  unchanged  for  several  days,  if  kept  cool.  Its 
application  is  principally  in  the  feeding  of  infants  when 
ordinary  milk  has  been  found  to  produce  undesirable  results. 
Freeman1  has  invented  a  pail  of  special  form  for  the  pas- 
teurization of  milk  in  bottles.  This  pail  is  filled  with  hot 

1  Medical  Record,  July  2,  1892,  and  August  4,  1894. 


68 


MANUAL    OF    BACTERIOLOGY. 


water  and  the  bottles  are  placed  in  it;  it  has  been  found  to 
keep  up  a  temperature  of  about  75°  C. 

The  Autoclave. — The  autoclave  is  an  instrument  de- 
signed for  sterilization  by  steam  under  pressure.  It  was 
invented  in  France  but  is  now  used  extensively  in  all  parts 
of  the  world.  Steam  generated  at  the  ordinary  atmospheric 
pressure  is  much  less  destructive  to  bacteria,  and  especially 

Vic..    15. 


Autoclave. 

to  their  spores,  than  steam  in  the  autoclave  at  a  pressure  of 
an  additional  one-half  to  one  atmosphere;  the  steam  then 
reaches  a  temperature  of  about  110°  to  I2O°C.  Under 
these  conditions  culture-media  may  be  sufficiently  sterilized 
in  the  autoclave  in  fifteen  minutes,  and  at  a  single  steriliza- 
tion. The  autoclave  consists  of  a  metal  cylinder  with  a 


STERILIZATION. 


69 


FIG.  i 6. 


movable  top,  which  is  fastened  down  tightly  during  sterili- 
zation. It  is  furnished  with  a  thermometer,  a  pressure- 
gauge,  and  a  safety-valve  which  allows  the  steam  to  escape 
if  too  high  a  pressure  is  attained.  Heat  is  furnished  by  a 
gas-burner  underneath.  The  lower  part  of  the  cylinder 
contains  water.  The  objects  to  be  sterilized  are  supported 
above  this  water  on  a  perforated  bottom  or  shelf. 

It  is  necessary  to  follow  certain  precautions  in  the  use  of 
the  autoclave,  especially  during  cooling.  The  apparatus 
must  not  be  opened  while  the  steam  contained  within  it  is 
still  under  pressure,  as  there  may  be  a 
sudden  evolution  of  steam  upon  the  re- 
moval of  the  pressure  which  may  blow 
the  media  out  of  their  tubes  and  flasks. 
The  apparatus  must,  therefore,  be  kept 
closed  until  the  gauge  shows  that  the 
atmospheric  pressure  is  as  great  as  the 
pressure  within,  or,  what  is  equivalent, 
until  the  temperature  has  fallen  to  100° 
C.  Gelatin,  especially,  may  be  dam- 
aged by  sterilization  with  the  autoclave, 
if  it  be  heated  too  long  or  to  too  high 
a  temperature. 

Sterilization  by  Filtration. — Ordi- 
nary filters  are  useless  for  this  purpose, 
but  the  tubes  or  bougies  of  unglazed 
porcelain  devised  by  Pasteur  and  Cham- 
berland  are  effective  when  properly  em- 
ployed. The  Berkenfeld  filter  employs 
bougies  made  of  infusorial  earth,  and  its  pores  are  larger  than 
those  of  the  Pasteur  filter.  Both  of  these  are  made  in  several 
grades  according  to  the  coarseness  or  fineness  of  the  pores. 
The  coarser  of  these  filters  permit  the  passage  of  very  small 
bacteria.  Bacteria  of  average  size,  like  bacillus  coli  com- 


Kitasato  Filter. 


JO  MANUAL    OF    BACTERIOLOGY. 

munis,  may  grow  through  the  pores  in  the  walls  of  both  the 
Berkenfeld  and  Pasteur  filters  if  sufficient  nutrient  material 
is  present  to  permit  of  their  multiplication.1 

Filters  of  these  kinds  are  widely  used  for  water,  and  will 
be  spoken  of  in  connection  with  the  chapter  on  water.  Simi- 
lar tubes  are  employed  for  the  filtration  of  certain  organic 
nutrient  media  whose  ingredients  would  be  damaged  by 
sterilization  with  heat,  chiefly  extracts  of  organs,  such  as 
the  thymus  gland.  The  soluble  "  toxins  "  of  bacteria  may 
be  obtained  by  filtration  of  fluid-cultures  through  such  tubes, 
which  remove  the  bacteria  (Fig.  16).  These  fluids  usually 
filter  very  slowly,  and  filtration  will  have  to  be  assisted  by 
some  form  of  vacuum-pump ;  usually  the  filter-pump,  which 
is  used  in  connection  with  a  stream  of  running  water,  is 
employed.  Compressed  air  or  carbonic  acid  may  be'  used  to 
assist  in  forcing  fluids  through  the  filter.  The  filter 
bougies,  the  flasks  and  all  parts  of  the  apparatus  must,  of 
course,  be  sterilized  by  heat  before  and  after  using. 

1  Wherry,  Journal  of  Medical  Research.  Vol.  VIII.,  1902. 


CULTURE-MEDIA.  7 1 


CHAPTER    III. 

CULTURE-MEDIA. 

CULTURE-MEDIA  are  substances  in  which  bacteria  are 
artificially  cultivated.  The  number  of  such  substances  is 
very  large,  different  materials  being  suited  to  different 
purposes  and  to  different  kinds  of  bacteria.  The  most 
important  ones  are  nutrient  bouillon  or  beef-tea,  nutrient 
gelatin,  and  nutrient  agar-agar.  The  two  last  have  a  jelly- 
like  consistency,  owing  to  the  addition  of  a  gelatinizing 
substance,  but  otherwise  are  of  the  same  composition  as 
bouillon. 

Nutrient  Bouillon. 

Beef-extract   (such  as  Liebig's) 3  grams. 

Peptone,    pure    (Witte's)1 10  grams. 

Sodium  chloride    (common  salt) 5  grams. 

Water    i  liter. 

The  solid  ingredients  are  dissolved  in  water,  and  the 
mixture  is  boiled  for  a  few  minutes.  It  is  made  neutral  or 
very  faintly  alkaline  by  the  addition  of  a  solution  of  sodium 
hydroxide,  drop  by  drop,  the  reaction  being  tested  at  inter- 
vals with  litmus-paper.  The  bouillon  may  now  be  filtered 
through  filter-paper.  The  filter-paper  should  be  folded  and 
creased  as  is  done  by  pharmacists;  it  is  usually  placed  in  a 
glass  funnel,  and  should  be  moistened  with  water  before 
using.  After  filtration  the  medium  is  to  be  placed  in  prop- 
erly plugged  tubes  or  flasks,  and  is  to  be  sterilized  once  in 
the  autoclave,  or  in  the  steam  sterilizer  for  fifteen  minutes 

1  Commercial  "peptones"  are  mixtures  of  albumose  and  a  small 
amount  of  peptone. 


72  MANUAL    OF    BACTERIOLOGY. 

or  longer  on  each  of  three  consecutive  days.  When  pre- 
cipitates form,  they  are  usually  caused  by  a  too  alkaline 
reaction.  That  may  be  corrected  by  the  addition  of  a  little 
weak  hydrochloric  acid,  drop  by  drop,  testing  frequently 
with  litmus-paper. 

A  more  accurate  way  of  obtaining  the  proper  reaction  is  Schultz's 
method.  Take  of  the  bouillon  10  c.c. ;  add  a  few  drops  of  phenol- 
phthalein1  (alcoholic  solution  i  per  cent.)  ;  with  a  burette  add,  drop  by 
drop,  a  solution  of  caustic  soda  0.4  per  cent,  until  a  faint  red  color  ap- 
pears, which  indicates  the  beginning  of  the  alkaline  reaction.  This 
procedure  is  followed  with  three  samples.  The  amount  of  soda  solu- 
tion required  in  each  case  is  noted  and  the  average  taken.  If  now,  on 
the  average,  for  each  10  c.c.  of  bouillon  i  c.c.  of  soda  solution  needs 
to  be  added,  for  1,000  c.c.  of  bouillon  100  c.c.  of  the  soda  solution  must 
be  added;  only,  instead  of  adding  a  weak  soda  solution,  one-tenth  as 
much  is  taken  of  a  solution  ten  times  as  strong. 

Another  method  of  making  bouillon  is  to  use,  instead 
of  beef-extract,  500  grams  (one  pound)  of  finely  chopped, 
lean  beef,  which  is  placed  in  one  liter  of  water  and  kept 
on  ice  for  twenty-four  hours.  It  is  strained,  thoroughly 
cooked  to  coagulate  the  albumen  in  it,  filtered,  and  a  liter 
of  fluid  obtained,  adding  water  if  necessary.  The  peptone 
and  salt  are  then  added  and  the  medium  heated  to  dissolve 
them.  It  is  then  neutralized,  filtered,  and  sterilized.  Al- 
though bouillon  made  with  solid  beef-extract  is  convenient 
and  serviceable  for  most  purposes,  it  is  advisable  to  use 
fresh  meat  when  the  bouillon  is  to  be  employed  for  the 
development  of  bacterial  toxins.  Fresh  meat  should  also  be 
used  in  the  preparation  of  either  bouillon,  gelatin  or  agar- 
agar  when  new  species  of  bacteria  are  being  studied  for  pub- 
lication. 

1  In  neutralizing  an  acid  culture-medium  it  has  been  found  that  when 
the  medium  appears  to  be  neutral  or  slightly  alkaline  to  litmus,  it  may 
still  be  acid  if  phenolphthalein  be  employed  as  an  indicator.  Fuller, 
Journal  .-lincrican  Public  llcaltli  Association.  1895. 


CULTURE-MEDIA.  73 

In  both  of  these  cases  the  recommendations  of  the  American  Public 
Health  Association  should  be  followed.1 

These  also  advise  that  media  be  neutralized  by  titration. — 

The  following  solutions  are  required :  $  per  cent,  phenolphthalein  in  50 
per  cent  alcohol,  normal2  (£')  and  twentieth  normal  (|^)  solutions 
of  sodium  hydroxide  and  of  hydrochloric  acid. 

To  5  c.c.  of  bouillon  in  a  porcelain  evaporating  dish  add  45  c.c.  of  dis- 
tilled water ;  boil  three  minutes ;  add  I  c.c.  of  phenolphthalein  solution, 
and  proceed  with  the  titration  wrwle  still  hot.  As  the  reaction  will 
usually  be  found  acid,  add  from  a  burette  /^  sodium  hydroxide  solu- 
tion, stirring  constantly,  until  a  decided  pink  color  develops  in  the 
entire  solution.  The  color  reaction  indicates  the  more  or  less  arbi- 
trarily adopted  neutral  point.  Repeat  this  procedure  with  three  differ- 
ent portions  of  bouillon,  and  determine  the  average  amount  of  ^ 
sodium  hydroxide  required.  It  is  now  possible  to  calculate  the  amount 

1  See  the  Report  of  the  Committee  of  the  American  Public  Health 
Association  entitled  Procedures  Recommended  for  the  Study  of  Bac- 
teria. 1898.  Rumford  Press,  Concord,  N.  H. 

2A  normal  solution  of  any  substance  contains,  fn  a  liter,  as  many 
grams  of  the  substance  as  there  are  units  in  its  molecular  weight,  in 
case  it  contains  a  single  atom  of  replaceable  hydrogen.  If  it  has  two 
atoms  of  replaceable  hydrogen  the  number  of  grams  used  equals  the 
molecular  weight  divided  by  two ;  and  so  on.  Thus  the  molecular 
weight  of  sodium  hydroxide  is  40,  and  its  normal  solution  contains  40 
grams  of  sodium  hydroxide  in  a  liter.  It  is  not  expedient  to  prepare 
normal  solutions  of  sodium  hydroxide  by  weight.  For  convenience, 
crystallized  oxalic  acid  is  used  as  a  starting  point  in  making  normal 
solutions.  Its  molecular  weight,  including  a  molecule  of  water  of 
crystallization,  is  123.  As  it  is  a  dibasic  acid  (having  two  atoms  of 
replaceable  hydrogen),  half  of  this  weight,  or  62.5  grams,  per  liter,  is 
taken.  Any  y"  acid  solution  will  exactly  neutralize  an  equal  volume  of 
any  ~  alkaline  solution.  To  make  j"  sodium  hydroxide  solution,  add 
about  41  grams  of  pure  caustic  soda  to  a  liter  of  distilled  water.  Find 
the  amount  of  this  solution  needed  to  exactly  neutralize  I  c.c.  of  y 
solution  of  oxalic  acid;  this  amount  contains  the  quantity  of  sodium 
hydroxide  which  should  be  present  in  I  c.c.  of  a  normal  solution.  It 
is  now  possible  to  calculate  the  amount  of  distilled  water  to  be  added 
in  order  that  I  c.c.  of  the  sodium  hydroxide  solution  may  neutralize 
i  c.c.  of  the  £  solution  of  oxalic  acid.  With  an  f  solution  of  sodium 
hydroxide  as  a  standard,  an  ^  solution  of  hydrochloric  acid  may  be 
prepared.  Twentieth  normal  solutions  have  one-twentieth  the  strength 
of  normal  solutions. 


74  MANUAL    OF    BACTERIOLOGY. 

of  y  sodium  hydroxide  needed  to  neutralize  the  whole  quantity  of 
bouillon.  This  should  be  added.  The  bouillon  should  then  be  boiled 
for  ten  minutes,  and  again  titrated.  It  will  usually  be  found  acid. 
The  deficiency  should  be  corrected  by  adding  the  necessary  amount  of 
y  sodium  hydroxide.  It  should  be  boiled  again,  and  again  titrated, 
and  any  deficiency  made  good.  It  is  rarely  necessary  to  repeat  the 
process,  except  to  determine  that  the  neutral  point  has  been  reached. 
After  neutralizing  it  is  boiled  thirty  minutes  and  filtered.  Enough  y 
hydrochloric  acid  or  sodium  hydroxide  is  added  to  give  the  degree  of 
acidity  or  alkalinity  desired.  It  is  then  sterilized. 

An  acid  reaction  may  be  denoted  by  +,  an  alkaline  by  — .  The 
degree  of  acidity  or  alkalinity  may  be  indicated  by  the  amount  of  £ 
solution  required  to  render  the  medium  neutral  to  phenolphthalein,  thus 
+  1.5  signifies  that  a  medium  is  acid,  and  requires  1.5  per  cent,  of  y 
sodium  hydroxide  to  neutralize  it. 

A  reaction  of  +1.5  is  recommended  as  the  optimum.  There  is  much 
disagreement  as  to  what  reaction  is  most  favorable  for  the  growth  of 
the  majority  of  species  of  bacteria.  In  any  case  the  degree  of  reaction 
should  be  noted  in  descriptions. 

Bouillon  may  be  modified  by  the  addition  to  it  of  other 
substances,  the  most  important  of  which  are  glycerine  (6  per 
cent.)  and  sugars, — as  dextrose,1  saccharose  or  lactose  (i 
per  cent.).  It  is  better  to  sterilize  media  containing  sugars 
in  the  steam  sterilizer  by  the  fractional  method  than  in  the 
autoclave,  where  decomposition  of  the  sugars  may  occur. 

Dextrose-free  Bouillon. — Ordinary  bouillon  often  contains  some 
muscle-sugar,  which  is  objectionable  if  fermentation  tests  with  lactose 
or  saccharose  are  to  be  made.  To  secure  bouillon  free  of  sugar,  beef- 
infusion  is  prepared  from  fresh  meat,  and  is  inoculated  in  the  evening 
with  a  quantity  of  bacillus  coli  commnnis,  and  kept  in  the  incubator. 
Early  next  morning  it  is  boiled,  filtered,  peptone  and  salt  added,  and 
the  bouillon  is  prepared  as  usual.2 

Nutrient  Gelatin. 

Beef-extract    3  grams. 

Peptone    10  grams. 

Sodium    chloride 5  grams. 

Gelatin    (best   gold   label) 100 grams. 

\Yuter     i  liter. 

1  Dextrose  is  the  principal  ingredient  of  commercial  grape-sugar  and 
should  be  obtained  in  a  pure  condition. 

2  See  T.  Smith,  Journal  of  Experimental  Medicine,  Vol.  II.,  p.  546. 


CULTURE-MEDIA.  75 

Dissolve  the  ingredients  in  the  water,  stirring  actively 
to  prevent  burning  at  the  bottom.  It  is  best  to  conduct  the 
operations  in  granite-  or  enamel-ware  vessels  over  a  large 
Bunsen  or  rose-burner.  Neutralize  with  sodium  hydroxide 
solution  (see  page  71).  The  reaction  at  the  beginning  will 
usually  be  found  to  be  quite  acid.  Allow  the  mixture  to  cool 
until  below  60°  C,  and  add  the  whites  of  one  or  two  eggs 
which  have  been  beaten  up  with  a  little  water;  stir  in  thor- 
oughly. Heat  the  mixture  to  the  boiling-point;  stir  at  the 
bottom  to  prevent  burning  and  at  the  same  time  avoid  as  far 
as  possible  breaking  the  coagulum  of  egg-albumen  which 
forms  at  the  surface.  Boil  for  ten  minutes.  Filter  while 
hot.  The  nitration  may  be  done  through  folded  filter-paper 
which  has  been  moistened.  It  is  well  to  fasten  a  piece  of 
coarse  cheese-cloth  over  the  top  of  the  funnel  to  catch  the 
large  particles  of  coagulated  albumen.  Place  in  suitable 
tubes  or  flasks  plugged  with  cotton,  and  sterilize  once  in  the 
autoclave,  or,  preferably,  in  the  steam  sterilizer  for  fifteen 
minutes  on  each  of  three  consecutive  days.  Gelatin  is  in- 
jured by  too  prolonged  boiling  and  loses  its  solidifying 
qualities.  Neutralization  may  be  with  litmus  paper  or  by 
titration.  The  remarks  on  pages  72  to  74  with  regard  to 
the  use  of  fresh  beef  and  the  titration  method  for  the  prepa- 
ration of  bouillon  apply  equally  to  gelatin. 

Instead  of  filter-paper,  some  prefer  to  filter  through  sev- 
eral layers  of  absorbent  cotton  placed  inside  of  the  moist- 
ened glass  funnel,  the  top  of  which  is  covered  with  coarse 
cheese-cloth.  This  expedient  answers  very  well. 

If  the  product  appears  cloudy  after  it  has  been  sterilized, 
it  may  be  that  the  egg-albumen  was  incompletely  coagu- 
lated in  the  first  place  or  that  the  reaction  has  been  made 
too  alkaline.  In  any  case  it  will  be  desirable  to  melt  it 
and  filter  a  second  time,  correcting  the  reaction  with  hydro- 
chloric acid  if  necessary.  It  may  be  well  to  stir  in  another 
7 


76  MANUAL    OF    BACTERIOLOGY. 

egg  to  entangle  the  opaque  particles;  then  to  boil  a  second 
time  and  filter. 

The  medium  is  sometimes  modified  by  adding  to  it  other 
substances,  as  sugar,  glycerin,  etc.  The  solidifying  prop- 
erty of  the  gelatin  must  be  carefully  guarded,  and  too  much 
boiling  is  to  be  avoided.  Certain  bacteria,  it  will  be  found, 
have  the  property  of  causing  gelatin  to  become  fluid. 
Gelatin  melts  at  about  25°  C.  and  solidifies  at  about  10°  C. 
It  cannot  be  used  in  the  incubator,  where  it  would  liquefy 
at  the  temperature  of  38°  C.  In  hot  weather  it  may  be 
necessary  to  use  150  grams  of  dry  gelatin  to  the  liter. 
Nutrient  gelatin  is  usually  spoken  of  simply  as  "  gelatin." 

Nutrient  Agar-agar. — Agar-agar  (French,  gelose)  is  a 
kind  of  vegetable  gelatin  which  comes  from  the  southern 
and  eastern  coast  of  Asia.  It  melts  with  much  greater  diffi- 
culty than  gelatin. 

The  medium  is  not  quite  transparent.  The  finished  me- 
dium is  commonly  called  "  agar.'1 

Beef-extract    3  grams. 

Peptone     10  grams. 

Sodium    chloride 5  grams. 

Agar     10  grams. 

Water     I  liter. 

The  dry  agar,  cut  tine,  is  to  be  dissolved  in  water  over  a 
flame.  It  should  be  boiled  for  from  one-half  hour  to  two 
hours,  skimming  off  the  scum  which  forms  on  the  surface 
from  time  to  time.  The  beef-extract,  peptone  and  sodium 
chloride  are  dissolved  in  a  liter  of  water,  boiled  and  neu- 
tralized. Add  the  agar  now  in  solution  in  a  small  quantity 
of  water.  The  reaction  of  the  agar  alone  is  faintly  alka- 
line. Mix  thoroughly;  the  bulk  of  the  mixture  is  a  little 
more  than  a  liter,  and  should  be  reduced  to  a  liter  after  the 
subsequent  boiling.  Cool  to  about  60°  C. ;  stir  in  the  whites 


CULTURE-MEDIA.  77 

of  one  or  two  eggs  and  boil  thoroughly.  Avoid  breaking 
the  eoagulum  of  egg  which  is  designed  to  entangle  the  solid 
particles  that  make  the  medium  cloudy;  stir  at  the  bottom, 
however,  to  prevent  burning.  Filter  while  hot,  using  filter- 
paper  or  absorbent  cotton  covered  with  cheese-cloth.  The 
hot  water  funnel  originally  devised  for  the  filtration  of  agar 
is  not  necessary.  If  filtration  is  slow,  the  funnel  and  flask 
may  be  placed  inside  of  the  steam  sterilizer  and  kept  heated 
during  filtration.  The  medium  is  collected  in  suitable  flasks 
or  tubes  plugged  with  cotton,  and  sterilized  once  in  the  auto- 
clave or  in  the  ordinary  steam  sterilizer  for  fifteen  minutes 
on  each  of  three  consecutive  days.  As  agar  is  frequently 
used  for  smear-cultures  where  a  slanted  medium  is  desired, 
some  of  the  tubes  may  be  allowed  to  cool  in  a  slanting  posi- 
tion. It  is  not  well  to  keep  on  hand  many  tubes  which 
have  been  slanted,  as  the  medium  dries  more  rapidly.  Agar 
is  not  liquefied  by  bacteria  as  is  gelatin.  Its  solidifying 
qualities  are  impaired  somewhat  if  the  reaction  be  acid. 

The  remarks  on  pages  72  to  74  with  regard  to  the  use  of 
fresh  beef  and  the  titration  method  for  the  preparation  of 
bouillon  apply  equally  to  agar-agar. 

Glycerin-agar  is  used  extensively.  It  is  agar,  made  as 
above  directed,  to  which  6  per  cent,  of  glycerin  is  added 
before  sterilization.  It  is  very  useful  in  cultivating  the 
bacilli  of  tuberculosis  and  diphtheria. 

Sugar-cigar. — Before  sterilizing,  i  per  cent,  of  either  dex- 
trose, lactose,  saccharose,  or  other  sugars  may  be  added  to 
agar.  With  media  containing  sugar,  litmus  forms  a  useful 
indicator  of  the  production  of  acid.  Enough  tincture  of 
litmus  is  used  to  give  the  medium  a  blue  color  before  ster- 
ilization; the  litmus  is  somewhat  unstable  and  prone  to 
change  its  color  during  sterilization.  Neutral  red  may  also 
be  added  in  the  same  manner ;  its  color  is  said  to  be  changed 
by  certain  bacteria  and  not  by  others  (see  bacillus  of  typhoid 
fever,  and  bacillus  coli  communis,  Part  IV.). 


MANUAL    OF    BACTERIOLOGY. 


FIG.  17. 


Potato. — The  potatoes  are  washed,  a  slice  is  removed 
from  each  end,  and  with  an  apple-corer  or  cork-borer  a 
cylinder  is  cut  out.  This  cylinder  is  divided  diagonally  into 
two  pieces.  The  pieces  are  washed  in  running  water  for 
twelve  to  eighteen  hours.  They  are  placed  in 
test-tubes  containing  a  little  water  to  keep  the 
potato  moist,  and  are  supported  from  the  bot- 
tom on  a  piece  of  glass  tubing  about  i  to  2 
cm.  in  length  (or  on  cotton,  or  in  a  specially 
devised  form  of  tube  with  a  constriction  at  the 
bottom) .  The  tubes  are  plugged,  and  sterilized 
as  with  other  media.  Sterilization,  however, 
must  be  thorough  on  account  of  the  danger 
of  contamination  with  the  extremely  resistant 
spores  of  the  potato  bacillus.  Potato  is  best 
when  freshly  prepared;  it  is  likely  to  become 
dry  and  discolored  with  keeping.  It  is  a 
very  useful  medium ;  certain  growths  on  it,  like 
those  of  the  bacillus  of  typhoid  fever  or  of 
glanders,  and  those  of  chromogenic  bacteria, 
are  very  characteristic. 

Milk. — Milk  fresh  as  possible  is  placed  in 
a  covered  jar,   sterilized   for  fifteen  minutes, 

Tube    contain-     an(j    t]ien    k      t    Qn    jce    £Qr    twenty-four    hours, 
ing    Jrotato. 

At  the  end  of  that  time  the  middle  por- 
tion is  removed  by  means  of  a  siphon.  The  upper  and 
lower  layers  must  not  be  taken;  the  upper  part  contains 
cream,  and  the  lower  part  particles  of  dirt,  both  of  which  are 
to  be  avoided.  About  7  to  10  c.c.  are  to  be  run  into  each 
test-tube.  The  tube  is  plugged  with  cotton,  and  sterilized 
as  usual.  When  milk  is  contaminated  with  spores  of  the 
hay  or  potato  bacillus  it  is  sometimes  very  difficult  to  steril- 
ize, a  fact  of  much  importance  in  connection  with  the  feed- 
ing of  children,  where  the  fractional  method  of  steriliza- 
tion and  the  use  of  the  autoclave  are  impracticable. 


CULTURE-MEDIA.  79 

The  coagulation  of  milk,  which  is  accomplished  by  cer- 
tain bacteria,  is  a  very  valuable  differential  point.  A  little 
litmus  tincture  may  be  added  to  the  tubes  of  milk  before 
sterilizing,  until  they  acquire  a  blue  color,  to  indicate 
whether  or  not  acids  are  formed  by  the  bacteria  which  are 
afterwards  cultivated  in  the  milk. 

Dunham's  Peptone  Solution. 

Peptone    10  grams. 

Sodium    chloride 5  grams. 

Water    i  liter. 

Boil,  filter,  sterilize  in  the  usual  manner. 

Dunham's  solution  is  valuable  to  test  the  development  of 
indol  by  bacteria  (see  Part  II.,  Chapter  II.).  The  develop- 
ment of  acids  may  be  detected  after  the  addition  of  2  per 
cent,  of  rosolic  acid  solution  (.5  per  cent,  solution  in 
alcohol)  ;  alkaline  solutions  give  a  clear  rose-color  which 
disappears  in  the  presence  of  acids. 

Blood-serum. — The  blood  of  the  ox  or  cow  may  be  ob- 
tained easily  at  the  abattoir.  It  should  be  collected  in  a 
clean  jar.  When  it  has  coagulated,  the  clot  should  be 
separated  from  the  sides  of  the  jar  with  a  glass  rod.  It 
may  be  left  on  the  ice  for  from  twenty-four  to  forty-eight 
hours.  At  the  end  of  that  time  the  serum  will  have  sepa- 
rated from  the  clot  and  may  be  drawn  off  with  a  siphon 
into  tubes.  These  tubes  are  sterilized  for  the  first  time  in 
a  slanting  position  as  the  first  sterilization  coagulates  the 
serum.  The  coagulation  may  be  done  advantageously,  as 
advised  by  Councilman  and  Mallory,  in  the  hot-air  sterilizer 
at  a  temperature  below  the  boiling-point.  After  coagulation, 
sterilize  as  usual.  This  serum  makes  an  opaque  medium  of 
a  cream  color.  Blood-serum  may  be  sterilized  in  the  special 
form  of  sterilizer  devised  for  it.  A  clear  blood-serum  is  to 
be  obtained  by  sterilization  at  a  temperature  of  58°  C.  for 
one  hour,  on  each  of  six  days,  if  a  fluid  medium  is  desired, 


8O  MANUAL    OF    BACTERIOLOGY. 

or  of  75°  G.  on  each  of  four  days  if  the  serum  is  to  be  solidi- 
fied. In  the  latter  case  the  tubes  are  to  be  placed  in  an  in- 
clined position.  (  See  page  67. )  Opaque,  coagulated  blood- 
serum  has  most  of  the  advantages  of  the  clear  medium. 
Blood-serum  may  be  secured  from  small  animals  by  collect- 
ing blood  directly  from  the  vessels,  using  very  great  care  to 
obtain  the  blood  in  a  sterile  condition ;  and  the  serum  may 
be  separated  and  stored  in  a  fluid  state.  Human  blood- 
serum  is  sometimes  obtained  from  the  placental  blood,  some- 
times from  serous  pleural  transudates  or  from  hydrocele 
fluid.  The  preservation  of  blood-serum  is  sometimes  accom- 
plished with  chloroform,  of  which  i  per  cent,  is  to  be  added 
to  the  medium ;  in  this  manner  the  serum  may  be  preserved 
for  a  long  time.  It  may  be  divided  into  tubes,  solidified  and 
sterilized  as  required ;  the  chloroform  will  be  driven  off 
by  the  heat,  owing  to  its  volatility.  Blood-serum  media 
which  are  sterilized  at  low  temperatures  should  be  tested  for 
twenty-four  hours  in  the  incubator  to  prove  that  steriliza- 
tion has  been  effective;  if  it  has  not,  development  of  the 
contaminating  bacteria  will  take  place  and  be  visible  to  the 
eye. 

It  will  be  impossible  to  do  more  than  merely  mention 
some  of  the  most  important  of  the  other  culture-media. 

Lbffler's  blood-serum  consists  of  one  part  of  bouillon 
containing  i  per  cent,  of  glucose,  and  three  parts  of  blood- 
serum.  It  is  sterilized  like  ordinary  blood-serum.  It  is 
used  largely  for  the  cultivation  of  the  bacillus  of  diph- 
theria. 

Blood-scntm-agar  is  a  medium  made  with  considerable 
difficulty,  but  very  valuable  for  the  cultivation  of  the  gon- 
ococcus.  One  part  of  placental  blood-serum,  or  pleuritic 
serum,  or  hydrocele  fluid,  is  mixed  with  one  to  two  parts 
of  nutrient  agar  in  the  fluid  condition.  It  must  be  divided 
into  tubes  before  solidification.  Solidify  in  a  slanting  posi- 


CULTURE-MEDIA.  8l 

tion;  subsequently  sterilize  at  75°  C.  so  as  not  to  coagulate 
the  albumen  of  the  blood-serum.  The  nutrient  agar  in  this 
case  should  contain  2  per  cent,  of  dry  agar.  Another  ex- 
pedient has  also  been  to  smear  a  little  blood  over  the  sur- 
face of  a  tube  of  nutrient  agar — blood-agar — used  for  culti- 
vating the  bacillus  of  influenza.  Marmorek's  blood-serum 
is  supposed  to  assist  in  maintaining  the  very  evanescent 
virulence  of  the  streptococci ;  it  consists  of  bouillon  mixed 
with  human  blood-serum,  ass's  serum  or  horse's  serum. 

Guarnieris  medium  consists  of  a  mixture  of  gelatin  and 
agar. 

Media  containing  fat  were  employed  by  Sommaruga  to 
test  the  ability  of  bacteria  to  decompose  fats.  Clarified 
beef-suet  or  olive-oil  in  the  proportion  of  I  or  2  per  cent. 
is  added  to  gelatin  or  agar.  The  fat  must  be  mixed  with 
the  melted  medium;  it  is  to  be  shaken  and  then  rapidly 
cooled  in  a  freezing-mixture  after  the  last  sterilization. 

Fresh  eggs  in  their  shells  may  be  used  without  other 
preparation  than  washing  the  surface  thoroughly  with  bi- 
chloride of  mercury  solution;  or  after  sterilization  by  steam, 
which  of  course  coagulates  the  albumen.  The  egg  is  easily 
inoculated  through  a  small  opening  made  with  a  heated 
needle,  which  may  be  closed  afterward  with  collodion. 
Hueppe  recommended  eggs  closed  in  this  manner  for  the 
cultivation  of  anaerobic  bacteria.  Egg-albumen  has  been 
used  as  a  constituent  of  various  media.  Dorset1  states  that 
good  results  may  be  secured  when  eggs  are  used  as  a  cul- 
ture-medium for  tubercle  bacilli.  The  yolk  and  the  white 
are  mixed,  poured  into  tubes,  slanted,  coagulated,  and  ster- 
ilized. Just  before  using  pour  into  the  tube  a  few  drops  of 
sterile  distilled  water  to  moisten  the  medium. 

Bread-paste  (finely-divided  dry  bread,  mixed  with  water 
and  sterilized)  is  used  for  the  cultivation  of  moulds.  Sa- 
1  American  Medicine,  April  5,  1902. 


82 


MANUAL    OF    BACTERIOLOGY. 


FIG.    18. 


bouraud  recommends  the  following  for  the  cultivation  of 
the  trichophyton  fungus : 

Peptone    5  grams. 

Maltose  3.8  grams. 

Agar  1.3  grams. 

Water    100  c.c. 

Test-tubes. — Bacteria  are  generally  cultivated  in  test- 
tubes.  A  convenient  size  is  one  f  of  an  inch  in  diameter 
and  5  inches  in  length.  The  tubes 
should  be  of  a  heavier  glass  than  in 
those  used  for  ordinary  chemical  work. 
The  New  York  Board  of  Health,  and 
some  others,  use  a  tube  three  inches 
in  length  without  a  flange  for  the  cul- 
tivation of  the  diphtheria  bacillus  on 
Loffler's  blood-serum  mixture.  Test- 
tubes  should  be  thoroughly  cleaned  with 
a  swab  before  using;  they  should  be 
boiled  with  washing-soda,  rinsed, 
filled  with  hydrochloric  acid  solution, 
rinsed,  and  inverted  to  drain  away  the  fluid. 

Plugs  of  raw  cotton  or  cotton  batting  are  employed  as 
stoppers.  Some  prefer  absorbent  cotton,  but  it  is  likely  to 
become  soggy  after  exposure  to  steam.  The  plug  should  fit 
smoothly;  creases  and  cracks  around  the  edges  are  to  be 
avoided.  The  plug  should  be  tight  enough  to  sustain  the 
weight  of  the  tube  when  held  by  the  plug.  These  plugs  pre- 
vent bacteria  from  entering  or  leaving  the  tubes. 

Sterilization  of  Test-tubes. — The  tubes  are  to  be  steril- 
ized in  a  hot-air  sterilizer  for  one  hour,  at  a  temperature 
of  150°  C.  The  cotton  should  acquire  a  light  brown  color 
but  should  not  be  burned.  If  the  plugs  touch  the  sides  of 
the  sterilizer  or  lie  against  the  bottom  they  may  be  scorched. 
The  necessity  for  sterilization  of  the  tubes  before  filling 


Wire  Basket  for  Test- 
tubes. 


CULTURE-MEDIA.  83 

them  with  the  medium  has  been  questioned,  and  it  is  prob- 
ably unnecessary  as  far  as  the  preservation  of  the  culture- 
medium  is  concerned,  but  it  will  be  found  that  the  cotton 
plugs  fit  much  better  after  sterilization  with  dry  heat. 
During  this  and  subsequent  sterilizations  the  tubes  are  held 
in  a  wire  basket. 

Filling  of  the  Tubes. — A  special  funnel  closed  with  a 
stop-cock  for  filling  tubes  with  liquefied  media  is  often 
recommended.  They  may  readily  be  filled  with  an  ordi- 
nary funnel  of  small  size.  During  the  filling,  the  neck  of 
the  test-tube  where  it  comes  in  contact  with  the  cotton  must 
not  be  wet  with  the  medium.  Ordinarily  about  7  to  10  c.c. 
are  placed  in  a  test-tube.  For  Esmarch's  roll-tubes  a  some- 
what smaller  quantity  is  desirable. 

The  sterilization  of  tubes  containing  culture-media  is 
always  done  by  steam,  and  has  been  sufficiently  described. 
It  is  to  be  remembered  that  the  solidifying  power  of  gelatin 
is  impaired  by  too  prolonged  heating,  while  heating  is  less 
likely  to  damage  other  culture-media.  The  media  which 
are  sterilized  at  a  low  temperature  (70°  C.)  should  be  tested 
for  two  days  in  the  incubator  to  determine  whether  steriliza- 
tion has  been  effective.  It  is  the  universal  experience  in 
bacteriological  laboratories  that  occasionally  culture-media 
will  become  contaminated  with  extremely  resistant  spores 
which  fail  to  be  sterilized  by  the  ordinary  processes,  an 
occurrence  which  causes  great  annoyance  and  calls  for  the 
exercise  of  much  patience.  Sometimes,  also,  moulds  attach 
themselves  to  the  plugs,  especially  if  they  are  moist,  and  send 
their  filaments  down  through  the  cotton ;  finally,  having 
reached  the  lower  edge  of  the  cotton,  their  spores  may  fall 
upon  the  medium,  grow  there  and  ruin  it. 


84  MANUAL    OF    BACTERIOLOGY. 


CHAPTER    IV. 

THE  CULTIVATION    OF   BACTERIA. 

Inoculation  of  the  Tubes. — The  air  of  the  laboratory 
should  he  as  quiet  as  possible,  to  lessen  the  chances  of  con- 
tamination by  bacteria  clinging  to  particles  of  dust.  Avoid 
working  where  there  may  be  draughts  or  gusts  of  air  or 
near  an  open  window.  Spores  are  blown  from  the  surfaces 
of  moulds,  like  thistle-down,  and  are  constantly  being  wafted 
about  in  the  air.  Given  any  material  containing  bacteria, 
for  example  a  pure  culture  of  some  well-known  species,  a 
very  minute  portion  is  to  be  introduced  into  a  tube  contain- 
ing the  sterile  culture-medium.  The  introduction  is  effected 
with  a  straight  platinum  wire,  or  with  a  platinum  wire  loop. 
The  platinum  is  to  be  heated  red-hot  before  using,  and  then 
allowed  to  cool.  It  is  also  to  be  heated  red-hot  after  using. 
The  plug  of  the  test-tube  is  to  be  withdrawn,  twisting  it 
slightly,  taking  it  between  the  third  and  fourth  fingers  of 
the  left  hand,  with  the  part  that  projects  into  the  tube  point- 
ing- toward  the  back  of  the  hand.  It  must  not  be  allowed  to 
touch  any  object  while  the  inoculation  is  going  on.  Pass 
the  neck  of  the  tube  through  the  flame.  If  any  of  the  cotton 
adheres  to  the  neck  of  the  tube,  pull  the  cotton  away  with 
sterilized  forceps,  while  the  neck  of  the  tube  touches  the 
flame,  so  that  the  threads  of  cotton  may  be  burned  and  not 
fly  into  the  air  of  the  room.  The  tube  is  held  as  nearly 
horizontal  as  possible.  The  tube  is  to  be  held  in  the  left 
hand  between  the  thumb  and  forefinger,  the  tube  resting 
upon  the  palm,  and  the  neck  of  the  tube  pointing  upward 


THE    CULTIVATION    OF    BACTERIA.  85 

and  to  the  right.  When  two  tubes  are  being  used  at  the 
same  time,  as  is  often  necessary,  they  are  placed  side  by  side 
between  the  thumb  and  forefinger  of  the  left  hand.  The 
two  plugs  are  held  between  the  second  and  third  and  the 
third  and  fourth  fingers  of  the  left  hand,  respectively.  The 
wire  may  now,  be  passed  into  the  first  tube,  which  we  will 
suppose  to  hold  some  material  containing  bacteria,  and  a 
little  of  this  material  may  be  removed  on  the  tip  of  the  wire 
from  the  first  tube  to  the  second.  When  the  needle  is  intro- 
duced into  or  removed  from  either  tube  it  should  not  touch 
the  side  of  the  tube  at  any  point,  and  should  only  come  in 
contact  with  the  region  desired.  After  inoculation  of  the 
second  tube  has  been  effected  the  wire  is  to  be  heated  to  a 
red  heat  in  the  flame,  the  necks  of  the  tubes  are  to  be  passed 
through  the  flame,  and  the  plugs  are  to  be  returned  to  their 

FIG.  19. 


Manner  of  Holding  Tubes. 

respective  tubes.  When  the  wet  wire  is  to  be  sterilized  in  the 
flame  it  should  be  approached  to  the  flame  gradually,  so  as 
to  dry  the  material  on  it  before  burning  it,  in  order  to  avoid 
"sputtering"  (see  page  33).  It  is  well  from  the  start  to 
train  one's  self  to  sterilize  the  platinum  wire  every  time  it  is 
taken  from  the  table  and  before  it  is  laid  down  again.  The 
platinum  wire  loop  may  be  used  in  the  same  manner  as  the 
straight  wire,  especially  when  a  substance  containing  a  small 
number  of  bacteria  is  being  handled. 


86 


MANUAL    OF    BACTERIOLOGY. 


When  a  tube  of  gelatin  is  to  be  inoculated  the  wire  is 
usually  introduced  into  the  medium  vertically,  "  stab-cul- 
ture " ;  when  a  medium  with  a  slanted  surface  is  employed, 
as  agar,  potato  or  blood-serum,  the  needle  should  lightly 
streak  the  surface,  "smear-culture"  (Figs.  20  and  21). 

The  safety  and  success  of  this  method  of  inoculation  de- 
pend upon  a  principle  which  has  been  established  by  long 
and  repeated  observation,  namely,  that  bacteria  do  not  of 
themselves  leave  a  moist  surface.  They  should  not,  there- 


FIG.  20. 


FIG.  21. 


Stab-Culture. 

A    rubber    stopper    may    be 

used  to  prevent  drying, 

see  page  91. 


Smear-Culture. 

This  tube  shows  the  rubber 

cap  used  to   prevent 

drying. 


fore,  rise  from  the  surface  of  the  moist  culture-medium,  nor 
drop  from  the  needle  during  its  transit,  if  proper  care  be 
exercised.  They  may  be  thrown  into  the  air  if  the  needle 
be  allowed  to  sputter  in  the  flame. 


THE    CULTIVATION    OF    BACTERIA.  87 

If,  by  any  accident,  drops  of  infectious  material  should 
fall  upon  a  surface  like  the  table,  they  should  be  covered 
at  once  with  bichloride  of  mercury  solution  i-iooo.  A 
good  way  is  to  cover  the  spot  with  a  piece  of  blotting-paper 
wet  with  the  solution ;  place  a  bell- jar  over  it  and  leave  for 
several  hours.  If  infectious  material  should  reach  the  hands 
or  clothing,  they  should  be  thoroughly  soaked  in  the  bichlo- 
ride solution.  When  working  with  pathogenic  bacteria  it 
is  well  to  wash  the  hands  in  this  solution  and  with  soap 
and  water,  as  a  routine  procedure,  before  leaving  the  labor- 
atory. 

To  maintain  their  vitality  bacteria  need  to  be  transplanted 
from  one  tube  to  another  occasionally;  the  time  varies 
greatly  with  different  species.  Many  bacteria  grow  on  cul- 
ture-media with  difficulty  at  the  first  inoculation,  but  hav- 
ing become  accustomed  to  their  artificial  surroundings,  as 
it  were,  they  may  be  propagated  easily  afterward;  this  is 
especially  true  of  the  bacillus  tuberculosis. 

Some  bacteria  flourish  better  on  one  culture-medium  than 
another.  The  bacillus  tuberculosis  grows  best  on  blood- 
serum  and  glycerin-agar ;  the  bacillus  of  diphtheria  grows 
best  on  LofBer's  blood-serum;  the  gonococcus  on  human 
serum-agar. 

The  virulence  of  most  pathogenic  bacteria  becomes 
diminished  after  prolonged  cultivation  upon  media.  Some- 
times the  virulence  is  lost  very  quickly,  for  example,  the 
streptococcus  pyogenes  and  micrococcus  lanceolatus  of  pneu- 
monia. 

Incubators. — Many  bacteria  flourish  best  at  a  tempera- 
ture about  that  of  the  human  body,  38°  C.  Some  species 
will  grow  only  at  this  temperature.  The  pathogenic  bac- 
teria in  particular,  for  the  most  part,  thrive  best  at  a  point 
near  the  body  temperature. 

The  incubator  is  a  box  made  of  copper,  having  double 


88  MANUAL    OF    BACTERIOLOGY. 

walls,  the  space  between  the  two  being  filled  with  water. 
The  outer  surface  is  covered  with  some  non-conductor  of 
heat,  such  as  felt  or  asbestos.  At  one  side  is  a  door,  which 

Tic.  22. 


Incubator. 

is  also  double.  The  inner  door  is  of  K"^lss<  the  outer  door 
is  of  copper  covered  with  asbestos.  At  one  side  is  a  gauge 
which  indicates  the  level  at  which  the  water  stands  in  the 


THE    CULTIVATION    OF    BACTERIA. 


89 


water-jacket.  The  roof  is  perforated  with  several  holes, 
some  of  which  permit  the  circulation  of  the  air  in  the  air- 
chamber  inside  the  box;  some  of  them  enter  the  water- 
jacket.  A  thermometer  passes  through  one  of  these  holes 
into  the  interior  of  the  air-chamber,  and  often  another  into 
the  water  standing  in  the  water-jacket.  A  gas-regulator 
passes  through  another  hole,  and  is  immersed  in  the  water 

FIG.  23.  FIG.  24. 


Reichert's  Gas-regulator.  Mercurial  Gas-regulator,     a.  Cham- 

ber   containing    volatile    hydrocarbon. 
b.  Capillary  opening. 

standing  in  the  water-jacket.  There  are  various  forms  of  gas- 
regulators  more  or  less  complicated.  In  general  they  consist 
usually  of  a  tube  containing  mercury ;  into  this  tube  are  two 
openings,  one  for  the  entrance  and  the  other  for  the  exit  of 
gas.  The  gas  enters  through  a  small  tube,  which  is  cut  off 
diagonally  at  the  bottom,  and  which  projects  into  the  sur- 
face of  the  mercury.  Heating  the  water  in  the  water-jacket 


9o 


MANUAL    OF    BACTERIOLOGY. 


Fir,.  25. 


causes  expansion  of  the  mercury,  which  rises,  and,  little  by 
little,  cuts  off  the  inflow  of  gas  through  this  tube.  The 
flow  is  never  completely  cut  off,  as  there  is  a  capillary  open- 
ing in  the  tube  considerably  above  any  point  to  which  the 
mercury  could  possibly  rise,  which  will  always  allow  the 
flow  of  a  small  quantity  of  gas  (Fig.  24,  b).  This  diagram 
also  shows  a  modification  of  the  simple  form  of  regulator, 
in  the  shape  of  a  partition  which  divides  off  a  lower  cham- 
ber, which  contains  mercury  and  is  connected  with  the  upper 

part  by  a  glass  tube.  The  pur- 
pose is  to  make  use  of  the  elastic 
properties  of  some  volatile  fluid, 
like  ether,  which  floats  on  the  sur- 
face of  the  mercury  at  a.  The 
gas  coming  from  the  gas-regulator 
passes  to  a  Bunsen  burner,  which 
stands  underneath  the  incubator. 
This  burner  should  have  some 
kind  of  automatic  device  for  cut- 
ting off  the  flow  of  gas  in  case  it 
becomes  accidentally  extinguished 
by  a  sudden  draught  of  air  or 
from  any  other  cause.  The  auto- 
matic burner  invented  by  Koch 
is  an  ingenious,  simple  and  effec- 
tive device.  A  bar  of  metal 
stands  above  the  flame;  by  its 

expansion,  through  a  system  of  levers,  it  supports  a  weight ; 
the  weight  controls  a  gas-cock.  While  the  flame  is  burning 
the  expansion  of  the  metal  holds  the  weight  horizontally;  if 
the  flame  becomes  extinguished,  the  metal  contracts,  the 
weight  falls,  and  cuts  off  the  flow  of  gas.  Some  incon- 
venience will  arise  from  irregularities  in  the  flow  of  gas 


Koch  Automatic  Gas-burner. 


THE    CULTIVATION    OF    BACTERIA.  91 

from  the  main  supply-pipe.  Any  incubator  will  vary  a  little 
from  such  causes.  In  the  experience  of  the  writer,  natural 
gas  is  of  such  variable  pressure  as  to  be  entirely  useless. 
Fluctuations  of  the  temperature  within  the  incubator  depend 
very  largely  upon  the  external  temperature.  Therefore  the 
incubator  should,  as  far  as  is  practicable,  be  protected  from 
sudden  draughts  of  cold  air  and  should  be  kept  in  a  room 
having  as  equable  a  temperature  as  possible. 

Culture-tubes  which  are  being  kept  in  the  incubator  are 
likely  to  become  dry  if  their  stay  is  prolonged.  In  such 
cases  they  should  be  covered  with  rubber  caps,  tin-foil, 
sealing-wax,  paraffin,  or  some  other  device  to  prevent 
evaporation.  If  rubber  caps  are  used,  they  should  be  left 
in  i-iooo  bichloride  of  mercury  solution  for  an  hour,  and 
the  cotton  plugs  should  be  singed  in  the  flame,  before  put- 
ting them  on.  (Fig.  21.)  The  writer  prefers  rubber 
stoppers,  which  may  be  boiled  and  stored  in  bichloride  of 
mercury  solution.  Cut  the  cotton  plug  even  with  the  edge 
of  the  tube ;  singe  it  in  the  flame ;  push  it  into  the  tube  about 
i  cm. ;  and  insert  the  rubber  .stopper.  (Fig.  20.) 

CULTIVATION  OF  ANAEROBIC  BACTERIA. 

The  cultivation  of  anaerobic  bacteria  is  done  best  in  a 
medium  containing  i  to  2  per  cent,  of  dextrose.  The  tube 
should  contain  a  large  quantity  of  the  culture-medium. 
Just  before  using,  the  medium  should  be  boiled  for  a  few 
minutes.  Inoculate  the  tube  after  cooling,  but  while  the 
medium  is  fluid.  Anaerobes  may  be  cultivated  in  the 
closed  arm  of  the  fermentation-tube  (see  Fig.  46),  but  the 
opening  between  the  two  arms  of  the  tube  must  be  small. 

Buchner's  method  for  the  cultivation  of  anaerobes:  Into 
a  bottle  or  tube  which  can  be  tightly  stoppered,  pour  10  c.c. 


MANUAL    OF    BACTERIOLOGY. 


FIG.  26. 


of  a  6  per  cent,  solution  of  sodium  or  potassium  hydroxide, 
for  each  100  c.c.  of  air  contained  in  the  jar.  Add  one  gram 
of  pyrogallic  acid  for  each  10  c.c.  of  solution.  The  culture- 
tube  is  placed  inside  of  the 
larger  bottle  or  tube,  supported 
above  the  bottom,  and  the  stop- 
per, smeared  with  paraffin,  is 
inserted.  The  mixture  of  pyro- 
gallic acid  and  potassium  hy- 
droxide possesses  the  property 
of  absorbing  oxygen. 

ITright's  Modification  of 
BiicJiucr's  method:  The  tube  of 
culture-medium  is  to  be  plug- 
ged with  absorbent  cotton,  us- 
ing a  plug  of  large  size.  The 
culture-medium  is  inoculated 
in  the  usual  way.  The  plug 
is  cut  off  close  to  the  neck  of 
the  tube,  and  is  then  pushed 
into  the  tube  about  I  centi- 
meter. Now  allow  a  watery 
solution  of  pyrogallic  acid  to 
run  into  the  plug,  and  then  a 
watery  solution  of  sodium  or 
potassium  hydroxide.  Close 
quickly  and  tightly  with  a  rub- 
ber stopper.  Wright  recommends  that  the  first  solution 
be  freshly  made  and  consist  of  about  equal  volumes  of 
pyrogallic  acid  and  water,  and  that  the  second  solution  con- 
tain I  part  of  sodium  hydroxide  and  j  parts  of  water.  With 
6  inch  test-tubes,  :|  inch  diameter,  the  amounts  advised  are 
-  .1  c.C.  solution  of  pyrogallic  acid,  I  c.c.  solution  of  sodium 
hydroxide. 


Arrangement   of  Tubes    for   Cul- 
tivation  of   Anaerobes    by 
Buchner's   Method. 


THE    CULTIVATION    OF    BACTERIA. 


93 


FIG.  27. 


Cultivation  of  Anaerobic  Bacieria  under  Hydrogen: 
Method  of  Frank  el:  A  test-tube  containing  a  large  amount 
of  the  liquefied  culture-medium  is  closed  with  a  sterilized 
rubber  stopper,  through  which  pass  two  sterilized  glass 
tubes,  bent  above  the  stopper  at  a  right  angle.  One  of 
these  tubes  is  cut  off  just  underneath 
the  stopper,  and  the  other  is  long 
enough  to  project  nearly  to  the  bottom 
of  the  culture-tube.  The  horizontal 
projecting  parts  are  drawn  to  a  small 
caliber  at  some  point,  although  not 
quite  closed,  to  facilitate  sealing  later 
on.  Through  the  longer  of  these 
tubes  hydrogen  gas  is  passed  until  the 
atmosphere  inside  of  the  culture-tube 
is  pure  hydrogen,  entirely  free  from 
mixture  with  air.  The  horizontal 
parts  of  the  small  glass  tubes  project- 
ing from  the  stopper  are  then  sealed 
in  the  flame  at  the  places  where  they 
were  previously  drawn  out  to  a  small 
caliber,  and  the  tubes  are  thus  closed. 
(Fig.  27.) 

The  stopper  should  be  surrounded 
with  melted  paraffin, 
according  to  this  plan  may,  if  desired, be 
converted  into  an  Esmarch  roll-tube.  The  hydrogen  is  gen- 
erated according  to  the  common  method  with  pure  zinc  and 
pure  sulphuric  acid,  25  to  30  per  cent.  The  precautions 
advised  by  chemists  for  the  generation  of  hydrogen  must 
be  carefully  followed,  because  when  hydrogen  mixed  with 
oxygen  or  air  is  ignited  a  violent  and  disastrous  explosion 
may  occur. 

The  well-known  Kipp's  generator  may  be  used.     First 


A  tube  prepared  Cultivation  of  Anaerobes 

by   Frankel's   Method. 


94 


MANUAL    OF    BACTERIOLOGY. 


FIG.  28. 


let  the  reservoir  fill  with  hydrogen;  then  allow  its  contents 
to  escape.  This  should  be  repeated,  after  which  some  of 
the  hydrogen  may  be  collected  in  an  inverted  test-tube  under 
water.  When  this  sample  is  ignited,  it  should  burn  without 
any  explosion ;  otherwise  the  hydrogen  is  not  yet  ready  to 
use.  The  hydrogen  should  bubble  through  the  medium  five 

minutes  or  more. 

The  inconvenience  of 
sealing  the  tubes  in  the 
flame,  as  has  to  be  done  in 
Franker s  and  other  meth- 
ods for  cultivation  under 
hydrogen,  is  obviated  in 
Novy's  apparatus.  The 
tubes  or  plates  are  placed 
in  jars  through  which 
hydrogen  may "  be  con- 
ducted. The  stopper,  hav- 
ing been  smeared  pre- 
viously with  a  soft  wax, 
is  sealed  by  giving  it  one- 
fourth  of  a  turn. 
Novy's  Jar  for  the  Cultivation  There  have  been  vari- 

of  Anaerobes.  oug    Qther    kinds    Q£    appa_ 

ratus,  usually  complicated  and  expensive,  devised  for  the 
growth  of  plate-cultures  under  hydrogen. 

Other  expedients  for  the  cultivation  of  anaerobic  bacteria  are  less 
effective.  In  cases  where  a  very  deep  stab-culture  is  made  in  gelatin 
or  agar,  where  the  growth  appears  in  the  lower  part  of  the  tube  by 
preference,  it  is  supposed  to  be  anaerobic.  Koch  covered  part  of  the 
surface  of  a  gelatin  plate  with  a  bit  of  sterilized  mica  or  a  cover- 
glass;  bacteria  which  grew  beneath  this  plate  were  considered  to  be 
anaerobic.  Another  method  was  to  cover  the  surface  of  the  gelatin  in 
the  cultu/e-tube  with  sterilized  oil.  W.  H.  Park  has  recommended  a  mix- 
ture of  solid  paraffin  with  25  to  50  per  cent,  of  fluid  paraffin  or  albolene 
as  a  covering  for  the  surface  of  anaerobic  cultures.  This  mixture  has  a 


THE    CULTIVATION    OF    BACTERIA. 


95 


semi-solid  consistency,  and  does  not  retract  at  the  edges  on  cooling. 
The  paraffin  prevents  the  absorption  of  oxygen,  except  to  a  small 
extent  at  the  edges.  The  method  is  useful  for  large  quantities  of  cul- 
ture material,  as  in  flasks.  Esmarch  advised  making  roll-tubes,  and 
after  cooling  them  to  fill  them  with  a  liquefied  gelatin  cooled  down 

FIG.  29. 


An    Aerobic    Organism    (Potato    Bacillus)    which    will    not    grow    under    a 

cover-glass. 

to  near  the  point  of  solidification.  Hueppe  made  use  of  eggs  in  their 
shells.  The  eggshell  was  carefully  cleaned,  sterilized  with  a  solution 
of  bichloride  of  mercury,  washed  with  sterilized  water  and  wiped  dry 
with  sterilized  cotton.  The  end  of  the  eggshell  was  punctured  with  a 
hot  needle.  Through  the  opening  thus  made  the  inoculation  was  accom- 
plished. The  opening  was  closed  with  collodion. 


96  MANUAL    OF    BACTERIOLOGY. 


CHAPTER  V. 

CULTIVATION    OF    BACTERIA,    CONTINUED. 

Isolation  of  Bacteria. — In  order  to  study  any  kind  of 
bacteria  it  is  necessary  to  have  the  particular  species  sepa- 
rated from  other  sorts  with  which  it  may  be  mixed.  The 
earlier  bacteriologists  endeavored  to  separate  bacteria  of 
different  sorts  by  successive  transplantations  through  a  series 
of  tubes.  The  procedure  now  generally  used  for  this  pur- 
pose is  the  so-called  plate-method  of  Koch.  The  great 
progress  which  bacteriology  has  made  during  the  last  twenty 
years  is  largely  owing  to  this  invention. 

Pathogenic  bacteria  may  sometimes  be  isolated  through 
inoculations  into  animals.  Thus  an  animal  may  be  inocu- 
lated with  sputum  containing  tubercle  bacilli  mixed  with 
other  bacteria.  The  animal  may  die  of  tuberculosis,  and 
its  tissues  may  contain  tubercle  bacilli  in  pure  culture,  the 
other  bacteria  having  produced  no  important  effect. 

Still  another  method  which  is  occasionally  useful  is  to 
subject  the  mixture  of  bacteria  to  steam  for  a  few  minutes. 
If  it  contains  very  resistant  spores,  like  those  of  the  teta- 
nus bacillus  or  hay  bacillus,  they  may  be  expected  to  sur- 
vive, and  may  perhaps  be  propagated  in  pure  culture, 
everything  else  having  been  killed  by  the  steam. 

Plate-Cultures. — It  is  impossible  in  most  cases  to  dis- 
tinguish between  bacteria  of  different  varieties  by  micro- 
scopical examination  alone.  Bacteria  of  widely  different 
species  and  quite  unlike  one  another  in  their  properties  may 
present  similar  appearances  under  the  microscope.  The 
differences  which  they  exhibit  are  usually  apparent  when 


THE    CULTIVATION    OF    BACTERIA.  97 

they  are  grown  in  culture-media.  The  growth,  called  a 
colony,  which  results  from  the  multiplication  of  a  single 
bacterium,  is  in  many  cases  quite  characteristic  for  the 
species.  By  the  plate-method,  the  individual  bacteria  in  a 
mixture  are  separated  from  one  another  by  dilution.  They 
are  fixed  in  place  by  the  use  of  a  solid  medium.  They 
are  allowed  to  grow,  and  from  each  individual  there  forms 
a  colony.  It  is  usually  possible  to  distinguish  between  col- 
onies arising  from  different  species  when  it  was  not  possible 
to  distinguish  between  the  individual  bacteria  of  these 
species.  A  convenient  illustration  has  been  suggested  by 
Abbott.  A  number  of  seeds  of  different  sorts  may  appear 
very  much  alike,  and  considerable  difficulty  may  be  found 
in  distinguishing  one  from  another  with  the  eye.  Let 
them  be  sown,  however,  and  let  plants  develop  from  them, 
and  these  plants  will  easily  be  distinguished  from  one 
another.1 

Method  of  Making  Plate-cultures. — Melt  three  tubes  of 
gelatin  or  agar.  (There  is  some  difficulty  in  keeping  agar 
in  a  fluid  state  while  dilutions  are  being  made.  It  is  best  to 
have  some  form  of  water-bath  with  a  thermometer  for  the 
purpose.)  Let  the  liquefied  tubes  cool  to  40°  C.  Take  a 
small  portion  of  the  material  to  be  examined — pus,  for  ex- 
ample— and  introduce  it  with  a  sterilized  platinum  wire  or 
loop  into  one  of  the  tubes.  Stir  it  in  carefully.  Remove 
the  needle,  sterilize  it,  and  replace  the  plug.  Mix  the  ma- 
terial introduced  thoroughly  with  the  liquefied  culture- 
medium,  taking  care  not  to  wet  the  plug.  Now  remove 
the  plug  again,  and,  having  sterilized  the  platinum  wire, 
insert  it  into  the  liquefied  medium.  Carry  three  loopfuls  in 

1  It  must  be  understood  that  no  close  comparison  can  be  drawn  be- 
tween higher  plants,  which  simply  complete  the  development  of  parts 
potentially  present  in  the  seed,  and  colonies  of  bacteria,  which  are  aggre- 
gates of  individuals,  the  progeny  of  one  individual  of  the  same  kind. 


98  MANUAL    OF    BACTERIOLOGY. 

succession  from  this  tube,  which  is  No.  i ,  into  tube  No.  2 ; 
sterilize  the  needle;  replace  the  plugs;  mix  thoroughly, 
without  wetting  the  plug.  Carry  three  loopfuls  from  tube 
No.  2  into  tube  No.  3  in  the  same  manner.  The  original 
material  will  obviously  be  diluted  in  tube  No.  I,  more  in 
tube  No.  2,  and  still  more  in  tube  No.  3.  The  most  con- 
venient form  of  plate  is  that  known  as  a  Petri  dish,  a  small 
glass  dish  about  8  cm.  in  diameter  and  1.5  cm.  in  height, 
provided  with  a  cover  which  is  a  little  larger  but  of  the  same 

FIG.  30. 


Petri  Dish. 

form.  This  dish  should  be  cleaned  and  sterilized  for  an  hour 
in  a  hot-air  sterilizer  at  150°  C.  or  higher.  When  it  is  cool 
it  may  be  used. 

Such  dishes  having  previously  been  prepared,  the  con- 
tents of  tube  No.  i  are  poured  into  one  dish,  and  those  of 
tube  No.  2  into  another,  and  those  of  tube  No.  3  into  a 
third.  They  are  to  be  labeled  Nos.  i,  2,  and  3.1  In  pour- 
ing proceed  as  follows  :  remove  the  plug  of  tube  No.  i ;  heat 
the  neck  of  the  tube  in  the  flame ;  allow  it  to  cool,  holding  it 
in  a  nearly  horizontal  position.  When  the  tube  has  cooled, 
lift  the  cover  of  the  Petri  dish  a  little,  holding  it  over  the 
dish;  pour  the  contents  of  tube  No.  I  into  the  dish,  and 
replace  the  cover  of  the  dish.  The  interior  of  the  dish 
should  be  exposed  as  little  and  as  short  a  time  as  possible. 

1  The  labels  should  be  moistened  with  the  finger,  which  has  been  dipped 
in  water.  They  should  not  be  licked  with  the  tongue.  While  working 
in  the  bacteriological  laboratory  it  is  best  to  make  it  a  rule  that  no  ob- 
ject is  to  be  put  in  the  mouth. 


. 


FIG.  31.     Dilution-cultures  in  Esmarch  Roll-tubes. 


FIG.  32.     Appearance  of  Colonies  on  Gelatin  in  Petri  Dish. 


THE   CULTIVATION    OF   BACTERIA.  99 

Tubes  Nos.  2  and  3  are  to  be  treated  in  the  same  manner. 
Burn  the  plugs,  and  fill  the  empty  tubes  with  5  per  cent. 
solution  of  carbolic  acid.  They  should  be  sterilized  for  an 
hour  in  the  steam  sterilizer  on  each  of  three  days. 

The  culture-medium  in  the  Petri  dish  will  soon  solidify. 
Colonies  develop  usually  in  from  one  to  two  days.  In 
plate  No.  i  they  will  be  very  numerous,  in  plate  No.  2  less 
numerous,  and  in  plate  No.  3  still  less  numerous.  Where 
the  "number  is  small  the  colonies  will  be  widely  separated 
and  can  readily  be  studied.  They  may  be  examined  with 
a  hand-lens,  or  the  entire  dish  may  be  placed  on  the  stage 
of  the  microscope  and  the  colonies  be  inspected  with  the 
low  power.  The  iris  diaphragm  should  be  partly  closed 
and  the  concave  mirror  should  be  used.  Dilution-cultures 
prepared  as  described  in  the  next  paragraph,  where  the 
principle  is  the  same,  are  shown  in  Fig.  31.  In  tube  No. 
i  the  colonies  are  so  numerous  as  to  look  like  fine  white 
dust.  In  tubes  2  and  3  they  become  less  numerous  and 
larger. 

Esmarch's  Roll-tubes. — Use  liquefied  gelatin  or  agar. 
The  dilutions  in  tubes  i,  2  and  3  are  made  as  above. 
Tubes  containing  a  rather  small  amount  of  the  culture- 
medium  are  more  convenient.  A  block  of  ice  should  be  at 
hand,  and,  with  a  tube  filled  with  hot  water  and  lying 
horizontally,  a  hollow  of  the  size  of  the  test-tube  should  be 
melted  on  the  upper  surface  of  the  ice.  In  this  hollow  place 
the  tube  of  liquefied  gelatin  or  agar;  roll  it  rapidly  with 
the  hand,  taking  care  that  the  culture-medium  does  not 
run  toward  the  neck  as  far  as  the  cotton  plug.  The  medium 
is  spread  in  a  uniform  manner  around  the  inside  of  the  tube, 
where  it  becomes  solidified.  Gelatin  roll-tubes  must  be 
kept  in  a  place  so  cool  that  there  is  no  danger  of  their 
melting;  in  handling  them  they  are  to  be  held  near  the 
neck,  so  that  the  warmth  of  the  hand  may  not  melt  the 

9 


100 


MANUAL    OF    BACTERIOLOGY. 


gelatin.  Agar  roll-tubes  should  be  kept  in  a  position  a 
little  inclined  from  the  horizontal,  with  the  neck  up,  for 
twenty-four  hours,  so  that  the  agar  may  stick  to  the  wall 
of  the  tube. 

By  the  plate-method  as  originally  devised  by  Koch,  instead  of  using 
Petri  dishes,  the  gelatin  was  poured  upon  a  sterile  plate  of  glass.  This 
plate  of  glass  was  laid  on  another  larger  plate  of  glass,  which  formed 
a  cover  for  a  dish  of  ice-water,  the  whole  being  provided  with  a  leveling 


FIG.  33. 


Manner  of  Making   Esmarch    Roll-tube. 

apparatus.  The  plate  was  kept  perfectly  level  until  it  had  solidified, 
which  took  place  rapidly  on  the  cold  surface.  The  glass  plates  were 
placed  on  little  benches  enclosed  within  a  sterile  chamber.  The  more 
convenient  Petri  dish  has  displaced  the  original  glass  plate  to  a  large 
extent. 

The  isolation  of  bacteria  may  sometimes  be  effected  by 
drawing  a  platinum  wire  containing  material  to  be  exam- 
ined rapidly  over  the  surface  of  a  Petri  dish  containing 
solid  gelatin  or  agar;  or  over  the  surface  of  the  slanted 
culture-medium  in  a  test-tube;  or  by  drawing  it  over  the 
surface  of  the  medium  in  one  test-tube,  then,  without  steril- 


THE    CULTIVATION    OF    BACTERIA.  IOI 

izing,  over  the  surface  of  another,  perhaps  over  several  in 
succession. 

Appearance  of  the  Colonies. — The  colonies  obtained  in 
the  Petri  dishes  or  roll-tubes  (Fig.  32)  may  be  studied 
with  a  hand-lens  or  with  a  low  power  microscope.  In  the 
latter  case,  use  the  concave  mirror  with  the  iris  diaphragm 
partly  closed.  The  colonies  present  various  appearances. 
Some  of  them  are  white,  some  colored;  some  are  quite 
transparent  and  others  are  opaque;  some  are  round,  some 
are  irregular  in  outline;  some  have  a  smooth  surface,  others 
appear  granular,  and  others  present  a  radial  striation. 
Surface  colonies  often  present  different  appearances  from 
those  occurring  more  deeply.  Surface  colonies  are  likely 
to  be  broad,  flat  and  spreading.  If  the  colony  consists  of 
bacteria  which  have  the  property  of  liquefying  gelatin,  a 
little  funnel-shaped  pit  or  depression  forms  at  the  site  of 
the  colony.  The  appearance  of  colonies  may  be  of  great 
assistance  in  determining  the  character  of  doubtful  species. 
The  appearance  in  gelatin  plates  of  the  colonies  of  the 
spirillum  of  Asiatic  cholera,  for  instance,  is  one  of  the  most 
characteristic  manifestations  of  this  organism. 

Pure  Cultures. — From  these  colonies  pure  cultures  may 
be  obtained  by  what  is  called  "  fishing."  Select  a  colony 
from  which  cultures  are  to  be  made;  touch  it  lightly  with 
the  tip  of  a  sterilized  platinum  wire,  taking  great  care  not 
to  touch  the  medium  at  any  other  point.  Introduce  the 
wire  into  a  tube  of  gelatin.  Sterilize  the  wire  and  plug  the 
tube.  In  a  similar  manner,  and  from  the  same  colony,  in- 
oculate tubes  of  agar,  bouillon,  milk,  potato  and  blood- 
serum.  At  the  same  time  it  is  well  to  make  a  smear  prepa- 
ration from  the  colony  and  to  stain  with  one  of  the  aniline 
dyes  so  as  to  determine  the  morphology  of  the  bacteria. 
The  growths  which  take  place  in  the  tubes  should  contain 
one  and  the  same  kind  of  bacteria.  As  seen  under  the  mi- 


T02  MANUAL    OF    BACTERIOLOGY. 

croscope  their  bacteria  should  have  the  same  general  form 
and  appearance  as  those  seen  in  the  colony  from  which 
they  were  derived.  This  will  be  the  case,  provided  the 
colony  has  resulted  from  the  development  of  a  single  bac- 
terium or  from  several  bacteria  of  the  same  kind.  Occa- 
sionally, however,  a  colony  will  develop  from  several  bac- 
teria which  may  not  all  be  alike.  In  that  case  a  pure 
culture  will  not  be  obtained,  and  the  process  of  plating 
may  have  to  be  repeated. 


I 
INOCULATION    OF    ANIMALS.  ICK 


CHAPTER    VI. 

INOCULATION    OF    ANIMALS. 

IN  the  study  of  pathogenic  bacteria,  the  inoculation  of 
animals  is  frequently  indispensable.  The  animals  most 
often  used  are  white  mice,  guinea-pigs,  rabbits  and  pigeons. 
Larger  animals  are  occasionally  employed  for  special  pur- 
poses. White  mice  may  be  kept  in  a  glass  jar  covered 
with  wire  netting.  They  may  be  fed  with  moistened  bread 
or  oats.  It  is  important  to  see  that  they  receive  drinking- 
water.  During  inoculation  the  mouse  must  be  kept  in 

FIG.  34. 


Mouse-holder. 

position  by  some  sort  of  mouse-holder,  or  may  be  held  by 
an  assistant,  who  takes  the  skin  at  the  back  of  the  neck 
between  his  fingers  and  at  the  same  time  holds  the  tail. 
The  hair  is  cut  off  from  the  skin  at  the  root  of  the  tail. 
A  small  V-shaped  opening  in  the  skin  is  made  with  scis- 
sors, and  a  stiff  sterilized  platinum  wire  is  passed  into  this 
opening,  separating  the  skin  from  the  muscles  for  some 
distance  so  as  to  make  a  pocket.  Into  this  pocket  the  ma- 
terial is  introduced  by  means  of  the  platinum  wire.  The 


IO4  MANUAL    OF    BACTERIOLOGY. 

wound  may  be  covered  with  collodion.  The  peritoneal 
p>1G  -  cavity  of  the  mouse  may  be  inoculated  with  a  fluid 
culture  introduced  with  a  sterile  hypodermic 
syringe. 

Guinea-pigs  and  rabbits,  after  inoculation,  are  to 
be  kept  in  cages  of  galvanized  iron  and  wire- 
netting.  The  bottom  may  conveniently  be  made  in 
the  form  of  a  movable  pan  which  permits  of  the 
disinfection  of  the  excreta.  Rabbits  and  guinea- 
pigs  may  be  fed  with  oats,  carrots,  cabbage,  grass 
and  the  like.  Guinea-pigs  and  rabbits  may  be  held 
by  an  assistant  or  tied  by  the  legs  upon  a  board. 
The  hair  over  a  small  portion  of  the  abdomen  is  cut 
away  and  a  short  incision  is  made  through  the  skin : 
a  pocket  is  produced  \vith  a  stiff  wire,  and  the  ma- 
terial inserted  with  a  sterile  platinum  wire.  The 
wound  may  be  covered  with  collodion.  Sutures 
may  be  used  if  the  wound  is  large.  Solid  sub- 
stances may  conveniently  be  introduced  by  placing 
them  in  a  sterile  glass  cannula,  which  is  pushed  to 
the  proper  situation  through  a  small  incision.  The 
substance  in  the  cannula  is  forced  out  of  it  with  a 
stiff  sterile  platinum  wire.  (Fig.  35.)  The  peri- 
toneal cavity  may  be  inoculated  with  a  previously 
sterilized  hypodermic  syringe,  or  an  incision  may 
be  made  which  reaches  to  the  peritoneal  cavity, 
into  which  the  desired  substance  may  be  introduced 
with  a  sterile  platinum  wire,  the  incision  being 
closed  with  sutures. 

Intravenous  inoculation  is  most  commonly  prac- 
ticed upon  rabbits.     A  small  vein  which  is  near  the 
posterior  margin  of  the  ear  of  the  rabbit  is  easily 
reached  from  the  dorsal  surface;    the  hypodermic 
needle  is  introduced  directly  into  this  vein.     In  making  a 


INOCULATION    OF    ANIMALS.  IO5 

hypodermic   injection,    the   needle   and   syringe   should    of 
course  be  sterilized  before  and  after  each  operation. 

Autopsies  upon  animals  should  be  held  as  soon  as  pos- 
sible after  death.  During  the  interval  the  body  should  be 
kept  in  the  ice-box.  The  autopsy  room  should  be  furnished 
with  screens  to  keep  out  flies,  so  that  they  may  not  light  on 
the  infected  animal.  The  animal  should  be  extended  on 
its  back  upon  a  board.  The  legs  may  be  fastened  with 
pins  or  tacks.  The  animal  should  be  handled  with  forceps 
as  far  as  possible,  and  after  beginning  the  autopsy  the 
fingers  should  not  touch  it.  If  the  fingers  come  in  contact 
with  infectious  matter,  disinfect  them  at  once.  Have  a 
basin  of  bichloride  of  mercury  solution  i-iooo  ready  for 
this  purpose.  Knives,  scissors,  platinum  wires  and  forceps 
should  be  sterilized  in  the  flame  before  and  after  each  ma- 
nipulation. Be  prepared  to  make  smear  preparations  on 
cover-glasses,  and  to  inoculate  tubes  of  gelatin,  agar  and 
other  media  as  desired.  Moisten  the  hairs  over  the  thorax 
and  abdomen  with  bichloride  of  mercury  solution  i-iooo, 
to  prevent  them  from  being  carried  into  the  air.  Make  an 
incision,  passing  through  the  skin  from  the  sternum  to  the 
pubis  along  the  thorax  and  abdomen,  and  diagonal  incisions 
extending  down  the  fore  and  hind  legs.  Dissect  away  the 
skin  from  the  thorax,  abdomen,  and  upper  parts  of  the  legs. 
With  a  knife  heated  in  the  flame,  sear  a  broad  line  extend- 
ing down  the  middle  of  the  abdomen.  Through  this  burned 
surface  make  an  incision  through  the  muscles  of  the  ab- 
domen. In  a  similar  manner  make  a  transverse  incision 
across  the  middle  of  the  abdomen  through  a  burned  sur- 
face. Cultures  should  be  made  from  the  peritoneal  cavity, 
and  smears  upon  cover-glasses  prepared,  which  are  after- 
wards to  be  stained.  With  a  hot  knife,  scorch  a  small  area 
on  the  surface  of  the  liver;  through  this  surface  enter  the 
liver  with  a  sterilized  platinum  wire,  and  with  the  material 


IO6  MANUAL    OF    BACTERIOLOGY. 

withdrawn  inoculate  the  tubes;  also  make  cover-glass  prep- 
arations. In  the  same  manner  inoculate  tubes  and  make 
cover-glass  preparations  from  the  spleen,  the  kidneys,  the 
pleural  cavity,  the  pericardial  cavity,  the  lungs,  and  the 
blood  inside  the  heart.  All  incisions  are  to  be  made  through 
the  burned  surfaces,  and  all  material  collected  for  inocula- 
tion is  to  be  obtained  through  burned  surfaces.  In  steriliz- 
ing the  instruments  in  the  flame  avoid  sputtering,  especially 
when  they  become  covered  with  oil  from  adipose  tissue. 
Pieces  of  lung,  liver,  spleen,  kidney  and  other  organs,  as 
may  be  indicated,  should  be  placed  in  95  per  cent,  alcohol 
for  fixation  and  hardening.  The  animal  and  the  board  on 
which  it  was  extended  should  be  covered  with  bichloride 
of  mercury  solution  i-iooo,  and  afterwards  burned.  The 
cage  or  jar  and  the  instruments,  dishes  and  towels  used 
should  be  sterilized  by  steam.  The  hands  of  the  operator 
should  be  washed  thoroughly  with  soap  and  water  and  with 
a  i-iooo  solution  of  bichloride  of  mercury. 

FIG.  36. 


Method  of  Making  Collodion  Capsules.     (After  McCrae.) 

Collodion  Capsules. — Bacteria  may  be  cultivated  in  the 
living  body  of  an  animal,  without  infecting  the  animal, 
when  they  are  enclosed  in  collodion  capsules.  Their  soluble 
products  are  able  to  diffuse  through  the  collodion,  while  the 
animal's  fluid  may  pass  into  the  sac  to  nourish  them.  These 
capsules  were  originally  made  by  dipping  the  round  end  of 
a  glass  rod  into  collodion  repeatedly.  McCrae's  method1 
is  easier  and  more  satisfactory.  (Fig.  36.) 

A  piece  of  glass  tubing  is  taken,  and  a  narrow  neck  drawn  on  it  near 
one  end.  This  end  of  the  tube  is  rounded  in  the  flame,  and  the  body 

'Journal  of  Experimental  Medicine,  Vol.  V.,  p.  635. 


INOCULATION    OF    ANIMALS.  IO/ 

of  a  gelatin  capsule  is  fitted  over  it,  while  still  warm,  so  that  the 
gelatin  may  adhere  to  the  glass.  The  capsule  is  now  dipped  into  3  per 
cent  collodion,  covering  the  gelatin  and  part  of  the  glass.  It  is  allowed 
to  dry  a  few  minutes,  and  is  dipped  again.  In  all  two  or  three  coatings 
may  be  given.  The  capsule  is  filled  with  water  and  boiled  in  a  test- 
tube  with  water.  The  melted  gelatin  is  removed  with  a  fine  pipette. 
The  capsule  is  partly  filled  with  water  or  broth  and  sterilized.  The 
capsule  may  now  be  inoculated.  The  narrow  part  of  the  neck  must 
then  be  sealed  in  the  flame,  taking  care  that  the  neck  be  dry.  The 
sealed  capsule  should  be  placed  in  bouillon  for  twenty-four  hours.  No 
growth  should  occur  outside  the  capsule  if  it  is  tight.  It  may  now  be 
placed  in  the  peritoneum  of  an  animal. 


10 


IO8  MANUAL    OF    BACTERIOLOGY. 


CHAPTER    VII. 

COLLECTION   OF   MATERIAL. 

SAMPLES  of  water  or  milk  collected  in  sterilized  tubes  or 
bottles,  when  they  are  not  examined  immediately,  or  when 
they  are  to  be  transmitted  any  distance,  should  be  kept  on 
ice.  Specimens  of  sputum  may  be  collected  in  clean  bot- 
tles tightly  corked.  They  should  be  examined  as  soon  as 
possible.  Although  decomposition  appears  not  to  interfere 
with  the  staining  properties  of  the  tubercle  bacilli,  the  spu- 
tum should  be  fresh  in  order  that  the  other  bacteria  con- 
tained in  it  may  be  studied.  Therefore  it  should  be  free 
from  contamination  with  putrefactive  germs.  Valuable 
information  can  also  be  obtained  by  examination  of  spu- 
tum in  a  fresh  condition  before  staining  (see  also  page  44). 

Samples  of  urine  keep  better  after  the  addition  of  a  few 
crystals  of  thymol,  which  retards  the  fermentative  process, 
so  that  the  sedimentation  of  the  bacteria  and  of  other  solid 
matter  in  conical  vessels  is  facilitated,  although  that  pur- 
pose can  be  accomplished  at  once  by  the  centrifuge. 
Thymol  will  also  be  a  useful  addition,  as  far  as  a  bacterio- 
logical examination  is  concerned,  in  case  samples  of  urine 
are  to  be  sent  by  mail;  thymol  should  not  be  added  if  cul- 
tures are  to  be  made. 

Specimens  of  sputum,  pus  or  blood  may  be  collected  con- 
veniently in  the  form  of  thin  smears  upon  cover-glasses. 
The  smears  are  fixed  by  passing  through  the  flame  three 
times.  Smears  of  blood  are  prepared  as  follows :  Have 
two  perfectly  clean,  square  cover-glasses.  The  finger,  or 
the  lobe  of  the  ear,  having  been  carefully  washed  with 


COLLECTION    OF    MATERIAL. 

water,  alcohol,  and  ether,  is  punctured  with  a  sterilized 
needle,  and  a  small  drop  of  blood  issues  which  is  wiped 
away.  The  second  drop  of  blood  should  be  taken ;  it  should 
be  about  the  size  of  a  pin's  head.  No  pressure  should  be 
exerted  upon  the  skin.  This  drop  of  blood  is  placed  on  one 
of  the  cover-glasses.  The  other  cover-glass  is  laid  upon  the 
first,  both  being  handled  with  forceps.  The  drop  of  blood 
becomes  flattened  out  into  a  thin  film.  Immediately  and 
before  the  blood  has  had  time  to  coagulate  the  two  are 
slipped  or  slid  away  from  each  other  in  a  horizontal  plane, 
not  forcibly  pulled  apart.  The  blood,  therefore,  will  be 
spread  in  thin  films  on  the  cover-glasses.  It  is  best  to  place 
the  cover-glasses  so  that  one  does  not  cover  the  other  ex- 
actly, but  so  that  the  sides  of  the  one 
lie  diagonally  to  the  sides  of  the  other, 
although  their  centers  coincide  (Fig. 
37).  Films  of  blood  which  are  to  be 
examined  for  the  parasite  of  malaria  may 
be  prepared  in  this  manner.  Samples  of 
blood  to  be  used  for  the  serum  reaction  ^/ 

for  typhoid  fever  need  to  be  pretty  good- 

•       ij  r  ut       j         u-   u  u  Manner  of  Placing 

sized  drops  of  blood,  which  may  be  col-  cover-glasses  in  Mak- 
lected  on  cover-glasses  or  pieces  of  un-  ing  Films  of  Blood, 
sized  paper  and  allowed  to  dry.  To  (After  Cabot.) 
test  blood  by  culture  methods,  i  to  5  c.c.  may  be  drawn 
from  a  vein  during  life,  using  a  sterilized  hypodermic  syringe 
and  all  antiseptic  precautions.  The  blood  thus  taken  may 
then  be  used  for  cultures  in  various  ways.  A  good  method 
for  general  purposes  is  to  empty  the  syringe  quickly  into  a 
flask  holding  100  c.c.  or  more  of  bouillon  or  dextrose- 
bouillon.  The  mixture  of  blood  and  bouillon  should  be 
placed  in  the  incubator  for  one  to  two  days.  If  the  bacteria 
develop,  they  may  be  secured  in  pure  cultures  by  plating, 
and  may  be  studied  further,  as  the  occasion  requires. 


IIO  MANUAL    OF    BACTERIOLOGY. 

At  autopsies  on  human  subjects  plate-cultures  should  be 
made,  if  possible,  directly  from  the  organs.  In  all  cases 
organs  should  be  entered  by  the  platinum  wire  through 
burned  surfaces.  The  method  of  isolation  by  streaking  the 
platinum  wire  containing  the  material  under  examination 
lightly,  several  times,  over  the  surface  of  an  agar  plate,  will 
be  found  convenient.  At  the  same  time  smears  should  be 
made  from  the  organs  upon  cover-glasses  for  microscopical 
study,  and  portions  of  the  organs  should  be  saved  and  hard- 
ened in  alcohol. 

A  convenient  device  for  the  collection  of  infected  mate- 
rial is  a  stiff  wire  wound  with  a  pledget  of  absorbent  cotton 
at  one  end,  the  whole  sterilized  in  a  tube,  as  recommended 
by  Warren  for  collecting  pus  and  other  fluids  for  examina- 
tion, and  as  introduced  by  W.  H.  Park  for  the  collection 
of  material  from  the  throat  in  cases  of  suspected  diphtheria 
(Fig.  78). 

The  so-called  Sternberg  bulb  is  valuable  for  the  collec- 
tion of  fluid  materials  for  examination.  A  short  piece  of 

glass  tubing  is  taken;  at  one  end 
FIG.  38.  is  blown  a  bulb ;  the  other  end  is 

drawn  out  to  a  long,  fine  point. 
To  introduce  the  substance  into 
the  bulb,  the  expanded  end  is 
Sternberg  Bulb.  heated  in  the  flame ;  the  point  is 

broken  and  introduced  below  the 

surface  of  the  fluid  which  is  to  be  collected;  as  the  bulb 
cools,  the  air  in  it  contracts  and  draws  the  fluid  into  it. 
When  it  has  taken  up  as  much  as  it  will,  the  point  may 
again  be  closed  in  the  flame. 

When  infectious  material  is  to  be  transported,  it  should 
be  so  packed  that  breakage  or  leakage  is  impossible. 

Concerning  the  transmission  of  materials  containing  bac- 
teria in  the  mails,  the  ruling  of  the  post-office  department 
of  the  United  States,  March  2,  1900,  is  as  follows: 


COLLECTION    OF    MATERIAL.  Ill 

"That  the  order  of  the  Postmaster  General  of  December  27,  1897 
(Order  No.  677),  amending  Order  No.  88  of  February  5,  1896,  prescrib- 
ing the  conditions  under  which  specimens  of  diseased  tissues  may  be 
admitted  to  the  mails  is  hereby  further  modified  in  the  following 
manner : 

"  Specimens  of  diseased  tissues  may  be  admitted  to  the  mail  for  trans- 
mission to  United  States,  State,  or  municipal  laboratories,  only  when 
enclosed  in  mailing  packages  constructed  in  accordance  with  the  specifi- 
cations hereinafter  enumerated :  Liquid  cultures,  or  cultures  of  micro- 
organisms in  media  that  are  fluid  at  the  ordinary  temperature  (below 
45°  C.  or  113°  F.)  are  unmailable.  Such  specimens  may  be  sent  in 
media  that  remain  solid  at  ordinary  temperatures. 

"  Upon  the  outside  of  every  package  shall  be  written  or  printed  the 
words  '  Specimen  for  Bacteriological  Examination.  This  package  to  be 
treated  as  letter  mail.'  No  package  containing  diseased  tissue  shall  be 
delivered  to  any  representative  of  any  of  said  laboratories  until  a  permit 
shall  have  first  been  issued  by  the  Postmaster  General  certifying  that 
said  institution  has  been  found  to  be  entitled,  in  accordance  with  the 
requirements  of  this  regulation,  to  receive  such  specimens." 

The  regulation  includes  not  only  cultures  but  "  moist 
specimens  of  diseased  tissues."  The  specifications  prescrib- 
ing the  manner  of  packing,  which  are  minute  and  compli- 
cated, may  be  obtained  from  local  postmasters. 


112  MANUAL    OF    BACTERIOLOGY. 


CHAPTER    VIII. 

SYSTEMATIC  STUDY  OF  SPECIES  OF  BACTERIA.1 

IN  order  to  conduct  the  study  of  any  species  of  bacteria 
it  is  necessary  to  have  the  organism  isolated  in  a  pure  cul- 
ture by  the  plate-method,  or  by  some  other  method  already 
described.  Having  thus  obtained  the  organism  in  pure 
culture,  it  is  to  be  examined  with  reference  to  its  behavior 
in  certain  particulars.  It  is  well  for  the  beginner  to  study 
a  few  known  species  of  saprophytes  obtained  from  some 
reliable  laboratory  in  pure  culture.  The  points  which  are 
to  be  considered  can  be  illustrated  best  by  presenting  them 
in  tabular  form,  filling  out  the  items  of  the  table  for  a 
given  species  of  bacteria. 

1.  Name. 

2.  Habitat  or  source. 

3.  Morphology;  grouping,  as  in  chains  or  in  zoogloe^e. 

4.  Size. 

5.  Staining  proporties.     Behavior  by  Gram's  Method. 

6.  Capsule,  present  or  otherwise. 

7.  Spore  formation. 

8.  Motility,  flagella. 

Growth  on  culture-media. 

9.  Relation  of  growth  to  temperature. 

10.  Gelatin;  observe  whether  the  gelatin  is  liquefied  or 
not.  Colonies  in  gelatin  plates,  study  under  low 
power  of  microscope. 

1  For  the   identification   of   unknown   species   consult   "A    Manual   of 
Determinative  Bacteriology,"  by  Frederick  D.  Chester. 


SYSTEMATIC    STUDY    OF    SPECIES    OF    BACTERIA.         113 

11.  Agar.      Colonies    in   agar   plates,    study   under   low 

power  of  microscope. 

12.  Bouillon,  note  cloudiness,  pellicle,  or  precipitate. 

13.  Milk;   observe  whether  or  not  the  milk   is   coagu- 

lated and  subsequently  peptonized. 

14.  Production  of  gas  in  fermentation-tube  with  bouillon 

containing  sugar,  as  dextrose,  or  in  agar  with 
sugars. 

15.  Potato. 

1 6.  Blood-serum;  observe  whether  or  not  peptonization 

occurs. 

17.  Production  of  indol. 

1 8.  Pigment  formation. 

19.  Production  of  acid  or  alkali. 

20.  Relation  to  oxygen;  observe  whether  the  superficial 

or  the  deep  part  of  the  growth  is  the  more  luxu- 
riant in  stab-cultures;  use  anaerobic  methods  if 
necessary. 

21.  Pathogenesis. 

In  commencing  the  study  of  bacteriology  the  pupil  should 
try  the  common  staining  methods  and  make  the  most  im- 
portant culture-media.  Having  culture-media  prepared,  it 
is  customary  to  study  a  number  of  species  of  non-pathogenic 
bacteria.  Notes  of  the  work  and  sketches  showing  the  mor- 
phology of  the  organisms  should  be  made.  It  is  well  to 
choose  species  which  have  properties  decidedly  different 
from  one  another.  The  micrococci,  bacilli  and  spirilla 
should  be  represented;  forms  that  are  motile  and  that  are 
not;  species  that  form  spores  and  others  that  do  not  form 
spores;  some  that  liquefy  gelatin  and  some  that  do  not. 
There  should  be  chromogenic  forms,  and  species  that  fer- 
ment dextrose,  and  that  produce  indol, — such  species  as 
some  of  the  sarcinse,  the  bacillus  coli  communis,  the  hay 
bacillus,  the  potato  bacillus,  bacillus  prodigiosus,  a  bacillus 


114  MANUAL    OF    BACTERIOLOGY. 

fluorescens  and  spirillum  rubrum.  It  is  well,  when  possible, 
to  obtain  material  directly  from  nature  rather  than  from 
laboratory  cultures.  This  may  readily  be  done  in  the  case 
of  the  hay  bacillus  and  the  potato  bacillus.  Fecal  matter 
may  be  spread  on  gelatin  plates  and  the  bacillus  coli  com- 
munis  obtained  in  pure  culture.  Fluorescing  bacilli  are  very 
common  in  water.  Large  spirilla  are  often  found  in  swamp 
water.  Some  organisms  like  spirillum  rubrum  can  only  be 
had  from  laboratory  cultures.  The  growth  of  some  aerobic 
organism,  like  the  potato  bacillus,  may  be  tested  under  a 
cover-glass  (see  Fig.  29).  The  pyogenic  bacteria,  which 
can  easily  be  isolated  from  pus,  may  be  studied  in  this  con- 
nection with  great  advantage.  The  staphylococcus  pyogenes 
aureus  and  the  streptococcus  pyogenes  should  on  no  account 
be  omitted.  The  diplococcus  of  pneumonia  can  most  readily 
be  obtained  from  a  mouse  or  a  rabbit  which  has  died  with 
pneumococcus  infection.  Such  an  animal  can  best  be  in- 
fected by  subcutaneous  inoculation,  using  some  of  the  rusty 
sputum  of  a  case  of  lobar  pneumonia.  The  cultivation  of 
the  pneumococcus  will  be  found  to  present  difficulties  in 
classes  containing  large  numbers  of  students. 

Representative  forms  of  moulds  and  yeasts  should  be 
studied  at  the  same  time.  Moulds  are  easily  obtained  by 
exposing  some  nutrient  substance  to  the  air,  covering  it, 
and  allowing  cultures  to  develop ;  yeasts  will  probably  grow 
also.  Ordinary  brewer's  yeast  may  be  isolated  in  pure  cul- 
ture from  gelatin  plates.  Bacteriological  examinations  also 
should  be  made  of  air,  soil,  water  and  milk.  With  such 
simple  means,  all  the  important  properties  of  bacteria  may 
be  demonstrated. 

Experiments  in  sterilization  and  disinfection  as  described 
in  Chapter  VIII. ,  Part  II. ,  may  be  performed  with  the  bac- 
teria mentioned,  which  present  every  variety  of  resisting 
power  up  to  the  almost  incredible  toughness  of  the  spores  of 
the  hay  and  potato  bacilli.  The  efficiency  of  the  methods 


SYSTEMATIC    STUDY    OF    SPECIES    OF    BACTERIA.         115 

used  for  sterilizing  surgical  materials,  as  silk  and  catgut 
(Chapter  IX.,  Part  II.),  should  be  tested;  also,  of  the 
methods  for  disinfecting  the  hands;  if  possible,  of  the 
methods  for  disinfecting  rooms,  as  well. 

After  some  proficiency  has  been  acquired,  various  patho- 
genic bacteria  may  be  studied  as  the  circumstances  of  the 
case  require.  Much  judgment  has  to  be  used  in  allowing  stu- 
dents to  work  with  pathogenic  bacteria.  Anthrax,  glanders, 
tetanus,  cholera,  bubonic  plague,  Malta  fever,  and  diphtheria 
all  have  occurred  in  laboratory  workers  through  accidental 
infection,  sometimes  with  fatal  results.  The  various  rules 
for  the  management  of  the  platinum-wire,  hanging-drop 
slides  and  sputum  bottles,  and  for  the  handling  of  cultures 
and  other  infectious  materials  have  already  been  given 
(pages  33,  36,  44  and  84  to  88).  The  most  important  pre- 
caution, perhaps,  is  observance  of  the  rule  that  while  work- 
ing in  the  laboratory,  nothing  should  be  put  in  the  mouth. 
Cultures  should  never  be  carelessly  left  in  improper  places. 
Cultures  of  bacteria  should  be  thoroughly  sterilized  before 
the  tubes  are  cleaned.  The  writer  is  in  the  habit  of  having 
tubes  and  dishes  containing  pathogenic  bacteria  placed  in  the 
steam  sterilizer  for  an  hour  on  each  of  three  days,  and  of 
having  the  plugs  removed  and  burned  and  the  tubes  filled 
with  5  per  cent,  carbolic  acid  between  the  second  and  third 
sterilizations.  In  taking  these  measures,  the  same  kind  of 
reasoning  applies  as  that  which  induces  engineers  to  give 
bridges  from  four  to  six  times  the  strength  they  need  to 
bear  the  greatest  strain  likely  to  be  put  upon  them,  or  to 
make  the  boiler  of  a  steam  engine  strong  enough  to  bear 
six  times  the  greatest  pressure  which  it  is  expected  that 
the  steam  contained  in  it  will  exert. 


PART    II. 


CHAPTER   I. 

CLASSIFICATION ;    GENERAL    MORPHOLOGY    AND    PHYSIOLOGY 
OF  BACTERIA. 

THE  relationships  existing  between  bacteria  and  other 
kinds  of  organisms  are  not  perfectly  clear.  It  is  quite 
generally  conceded,  however,  that  bacteria  are  plants.  They 
show  affinities  with  both  the  lower  algse  and  the  lower  fungi, 
and  they  have  even  some  points  of  resemblance  with  certain 
of  the  protozoa.  On  account  of  their  extreme  smallness  it 
is  impossible  to  analyze  the  structure  of  the  individual  bac- 
teria and  to  contrast  the  structure  of  one  with  that  of 
another.  The  classification  cannot  therefore  be  established 
on  morphological  grounds  chiefly,  as  is  done  with  large 
animals  or  plants.  We  are  obliged  to  rely  also  upon  their 
growth  with  relation  to  the  presence  or  absence  of  oxygen 
and  to  temperature,  their  behavior  on  culture-media,  the 
appearances  of  the  growths,  and  the  production  of  certain 
substances  with  peculiar  chemical  reactions,  when  we  wish 
to  establish  the  points  of  difference  between  one  species  and 
another — all  of  which  is  extremely  unsatisfactory  and  prob- 
ably not  perfectly  reliable.  It  is  likely  that  forms  which  are 
now  considered  as  different  species  are  not  really  such  in  all 
cases,  and  also  that  different  species  may  now  be  included 
under  one  heading  as  a  single  species.  Notwithstanding  the 
unsatisfactory  condition  of  the  classification  of  bacteria,  it 
must  not  be  supposed  that  the  species  of  bacteria  are  not 

117 


Il8  MANUAL    OF    BACTERIOLOGY. 

permanent.  For  instance,  it  would  be  incorrect  to  imagine 
that  it  is  possible  for  the  micrococci  and  spirilla  to  become 
converted  into  species  of  bacilli,  or  for  the  bacilli  of  one 
species  to  be  transmuted  into  those  of  another.  This  does 
not  contradict  the  statement  that  we  may  frequently,  through 
erroneous  and  imperfect  information,  be  in  the  habit  of 
including  unlike  species  under  one  name,  or  of  classifying 
mere  varieties  of  one  species  as  entirely  different  species.  At 
present  the  simple  division  of  bacteria  into  three  great 
generic  groups  is  probably  as  good  as  any :  micro  cocci, 
spherical  forms;  bacilli,  rod-shaped  forms,  one  diameter 
being  in  excess  of  the  others;  spirilla,  twisted  like  a  cork- 
screw, making  long  spirals  or  simply  parts  of  spirals 
( comma-shaped  forms ) . 

Recent  investigations  indicate  that  several  species  of  bac- 
teria often  are  closely  related  to  one  another,  so  as  to  form 

FIG.  39. 


Staphylococci.   Streptococci.  Diplococci.  Tetrads.   Sarcinae. 

a  well-marked  group.  Such  a  group  is  constituted  by  the 
bacillus  of  typhoid  fever,  bacillus  coli  communis  and  similar 
forms.  The  spirillum  of  cholera  and  other  comma-shaped 
spirilla  resembling  it  may  be  held  to  constitute  another 
group.  Still  another  is  that  containing  the  tubercle 
bacillus  and  other  acid-proof  bacilli. 

The  micrococci  are  subdivided  into  Staphylococci,  where 
the  spheres  grow  in  clusters  like  a  bunch  of  grapes;  strep- 
tococci, where  they  are  arranged  in  long  rows  or  chains, 
like  a  string  of  beads ;  diplococci,  or  pairs  of  micrococci ; 
tetrads,  where  the  individual  spheres  are  grouped  in  fours; 
sarcincr,  where  they  are  grouped  in  eights,  making  the  out- 
line of  a  cube,  resembling  a  bale  or  package  tied  with  rope. 


MORPHOLOGY    AND    PHYSIOLOGY    OF    BACTERIA.         1 19 

The  bacilli  are  not  usually  subdivided  in  this  manner, 
although  their  forms  vary  considerably.  The  ends  are 
sometimes  square,  sometimes  round.  Sometimes  they  are 
very  short.  Sometimes  they  grow  in  longer,  thread-like 
forms,  in  which,  however,  the  transverse  markings  which 

FIG.  40. 


»«  -& 


Bacilli  of  Various  Forms. 

indicate  the  outlines  of  the  individual  bacilli  can  generally 
be  seen,  and  which  resemble  a  bamboo  rod.  Short  oval 
bacilli  may  look  exceedingly  like  micrococci.  Bacilli  with 
rounded  extremities,  placed  end  to  end,  look  like  strings  of 
sausages.  Under  exceptional  circumstances,  branching 
forms  of  the  bacilli  of  diphtheria,  tuberculosis,  glanders  and 
bubonic  plague  and  various  other  species  have  been  en- 
countered.1 

The  word  ce  bacterium  "  was  formerly  used  to  designate 
short  bacilli  which  generally  formed  no  spores,  while  the 

FIG.  41. 


Spirilla  of  Various  Forms. 

word  bacilli  was  restricted  to  the  longer  forms  in  which 
spore  formation  occurred.  This  use  is  no  longer  common, 
although  not  rarely  the  name  bacterium  is  still  given  to  a 
species — for  instance,  bacterium  coli  commune. 

1  See  Hill,  Journal  of  Medical  Research,  Vol.   VII.,  January,   1902; 
Loeb,  ibid.,  Vol.  VIII.,  1902. 


I2O  MANUAL    OF    BACTERIOLOGY. 

Spirilla  present  a  very  great  variety  of  form.  The  short 
{f  comma-shaped  bacilli "  are  only  parts,  at  most,  of  spirals, 
although  the  microbes  of  cholera  do  sometimes  form  long 
spirals.  On  the  other  hand,  there  are  among  spirilla  large 
and  long  sinuous  figures  which  present  most  remarkable 
pictures  under  the  microscope;  for  example,  the  spirillum 
of  relapsing  fever.  Formerly  spirilla  without  very  marked 
windings  were  called  "  vibrios " ;  and  long,  wavy  forms 
with  corkscrew-like  windings  "  spirochcetcu " ;  and  the 
rigidly  spiral  forms  were  denominated  "  spirilla."  These 
definitions  have  for  the  most  part  lost  their  significance, 
although  the  names  still  linger  in  nomenclature. 

FIG.  42. 


Involution  Forms  of  the  Spirillum  of  Cholera.      (Van  Ermengem.) 

Besides  the  classification  already  mentioned,  bacteria  are 
sometimes  grouped  according  to  certain  other  qualities.  In 
general  botany,  saprophytes  are  plants  that  grow  on  decay- 
ing vegetable  matter.  In  a  bacteriological  sense,  sapro- 
phytes are  bacteria  which  grow  in  external  nature  on  dead 
organic  matter,  and  parasites  are  bacteria  which  exist  upon 
the  living  tissues  or  fluids  of  any  organism.  Nearly  synon- 
ymous with  the  above  words  are  those  which  do  not  and 
those  which  do  produce  disease,  or  non-pathogenic  and 


MORPHOLOGY   AND    PHYSIOLOGY   OF    BACTERIA,         121 

pathogenic.  The  adjectives  facultative,  or  optional,  and 
obligate,  or  strict,  are  used  to  qualify  the  above  terms  and 
many  others. 

Size. — Bacteria  vary  greatly  in  size.  The  micrococci  are 
usually  i  [J.  or  less  in  diameter.  The  short  diameters  of 
bacilli  and  spirilla  also  are  less  than  i  fJ.  as  a  rule,  while 
the  length  may  be  several  p.  .  The  anthrax  bacillus  (1.5  A* 
X  3  to  10  p)  and  the  spirillum  of  relapsing  fever  are  the 
largest  bacteria  known  to  be  pathogenic  to  man.  To  say 
that  a  micrococcus  is  i  p.  in  diameter  means  that  25,000 
end  to  end  would  make  a  line  I  inch  long.  It  has  been 
estimated  that  I  milligram  of  a  pure  culture  of  the  staphy- 
lococcus  pyogenes  aureus  contains  8,000,000,000  micro- 
cocci. 

There  is  good  reason  for  believing  that  organisms  exist, 
which  are  too  small  to  be  visible  with  the  most  powerful 
microscopes.  The  nature  of  these  organisms  is  not  known, 
but  it  is  not  improbable  that  some  of  them  are  bacteria. 
(See  pleuro-pneumonia  of  cattle,  etc.,  Part  II.,  Chapter  V.) 

In  stained  preparations  the  bodies  of  bacteria  frequently 
seem  to  be  homogeneous.  On  the  other  hand,  they  may 
exhibit  certain  spots  which  stain  more  intensely  than  others, 
the  stained  spots  alternating  with  clear  areas.  The  dark- 
staining  granules  may  take  a  slightly  different  shade  of  color 
from  the  rest  (metachromatic  granules,  Babes-Ernst 
bodies).  Somewhat  similar  appearances  may  result  from 
changes  in  the  density  of  the  protoplasm  of  bacteria,  leaving 
vacuoles  that  do  not  stain  (plasmolysis). 

In  old  cultures  bacteria  are  likely  to  show  deformed  and 
twisted  outlines  called  involution  forms.  It  is  not  uncom- 
mon for  bacteria  to  be  enclosed  in  a  kind  of  envelope  of 
some  clear  substance,  which  stains  with  difficulty  or  not  at 
all,  called  a  capsule.  The  paired  micrococci  of  pneumonia 
are  inclosed  in  such  capsules.  The  capsule  is  more  likely  to 


122  MANUAL    OF    BACTERIOLOGY. 

be  demonstrated  when  the  bacteria  are  obtained  from  the 
fluids  derived  from  an  animal's  body  than  when  they  have 
been  grown  artificially  in  culture-media.  A  zoogloea  is  a 
large  mass  of  bacteria  in  a  resting  condition  held  together 

by  a  mucilaginous  substance.  The 

F  IG.  43. 

composition  of  bacteria  varies  con- 


<*?>  I)  siderably   with    different    species. 

The  basis  aPPears  to  be  Proteid 
substance. 

T*16  multiplication  of  bacteria 

Bacteria  with  Capsules.          takes  Place  in  almost  all  CaSCS  by 

transverse  fission.   The  formation 

of  tetrads  or  sarcinae  from  micrococci  depends  upon  fission  in 
two  or  three  planes.  Repeated  fissions  of  micrococci  in  one 
plane  result  in  the  formation  of  streptococci.  Micrococci 
that  have  recently  divided  are  likely  to  be  somewhat  flat- 
tened. Multiplication  under  favorable  circumstances  may 
take  place  at  a  phenomenally  rapid  rate.  Bacilli  have  been 
observed  to  divide  in  twenty  minutes.  If  division  takes 
place  once  in  an  hour,  the  progeny  of  one  organism  at  the 
end  of  twenty- four  hours  will  be  16,777,216,  i.  e.,  (2  X  i)24- 
The  ordinary  form  of  reproduction  by  fission  is  called 
vegetative,  and  bacteria  that  are  multiplying  in  this  man- 
ner are  often  spoken  of  as  being  in  the  vegetative  condi- 
tion. 

FIG.  44. 


Or 

); 


Bacteria  with   Spores. 


Spores.  —  Under  certain  circumstances  the  reproduction 
of  bacteria  takes  place  by  means  of  bodies  called  spores. 


MORPHOLOGY    AND    PHYSIOLOGY    OF    BACTERIA.         123 

They  appear  in  a  typical  form  in  the  large  bacilli,  where, 
near  the  centers  of  the  bacilli,  highly  refracting,  shining 
spots  may  be  seen  which  are  found  to  stain  less  rapidly 
with  the  "aniline  dyes  than  the  rest  of  the  bacilli.  They 
are  not  to  be  confused  with  the  unstained  spots  described 
as  vacuoles.  On  account  of  their  being  formed  from  a 
part  of  the  interior  of  the  bacterium,  such  spores  are  called 
endogenous.  These  spores  are  found  mostly  in  the  bacilli, 
rarely  in  spirilla  and  micrococci.  They  are  what  is  meant 
when  the  word  spore  is  used  alone  without  qualification. 
The  existence  of  another  kind  of  spore,  described  as  form- 
ing from  the  whole  of  the  bacterium  (called  arthrospore) , 
is  doubtful.  At  all  events,  its  significance  is  not  at  present 
understood.  Spores  develop  generally,  though  not  always, 
under  adverse  conditions  of  various  kinds,  as  of  tempera- 
ture and  of  nutrition.  They  are  more  resistant  to  unfavor- 
able influences  of  all  sorts  than  are  the  fully  developed 
bacteria.  Spores  resist  drying,  light,  heat  and  chemical 
agents  to  a  remarkable  degree,  at  times. 

Anthrax  spores  are  said  to  have  been  found  which  could 
withstand  steam  for  twelve  minutes,  i-iooo  mercuric  chlo- 
ride for  nearly  three  days,  or  5  per  cent,  carbolic  acid  for 
more  than  forty  days.  The  greatest  resistance  is  displayed 
by  the  spores  of  some  of  the  saprophytic  bacteria,  particu- 
larly those  of  hay  and  potato,  which  are  sometimes  not 
destroyed  by  several  hours  of  steaming;  and  bacteria  are 
said  to  have  been  obtained  from  the  soil  which  resisted 
100°  C.  for  sixteen  hours.  When  cultivated  at  a  tempera- 
ture as  high  as  42°  C.  the  anthrax  bacillus  becomes  inca- 
pable of  forming  spores.  Spores  themselves  do  not  mul- 
tiply, nor  do  they  manifest  any  activity.  Spores  may  be 
located  at  the  center  of  the  bacillus,  or  nearly  at  one  end, 
when  the  end  of  the  bacillus  is  likely  to  enlarge,  making 
a  form  having  the  shape  of  a  drumstick,  as  takes  place 
ii 


124  MANUAL    OF    BACTERIOLOGY. 

with  tetanus  bacilli  (Fig.  44).  When  a  bacillus  assumes  a 
spindle  shape  on  account  of  having  the  middle  part  bulged 
through  the  formation  of  a  spore  it  is  called  a  clostridium. 
With  rare  exceptions,  a  single  bacillus  contains  but  one 
spore.  Under  favorable  conditions  the  spores  germinate,  as 
it  is  called,  and  develop  to  the  adult  form  of  the  organism. 
This  may  be  witnessed  in  hanging-drop  preparations. 

Motility. — Motility  is  rarely  exhibited  by  micrococci ; 
some  bacilli  possess  it  and  some  do  not;  while  nearly  all 
of  the  spirilla  are  motile.  The  phenomenon  is  observed  in 
the  hanging-drop.  The  degree  of  motility  is  variable,  being 

FIG.  45. 


Bacteria    showing    Flagella. 

sometimes  slight  and  sometimes  very  active.  When  seen 
under  a  high  power  the  little  particles  taken  from  a  cul- 
ture of  a  motile  organism  may  look  like  a  writhing  mass 
of  maggots  or  like  tadpoles  in  a  pool.  The  motility  is  most 
active  in  young  cultures.  The  movement  results  from  the 
vibration  of  little  processes,  or  flagella  (Fig.  45).  Of  these 
there  may  be  one  or  several.  They  may  be  placed  singly 
or  in  groups,  at  the  end,  or  scattered  around  the  sides.  They 
are  extremely  difficult  to  demonstrate  except  by  special  stain- 
ing methods,  which,  furthermore,  are  decidedly  capricious. 
After  the  flagella  have  been  stained,  the  bacteria  appear 
somewhat  larger  than  when  stained  by  the  ordinary 
methods.  The  flagella  upon  the  bacilli  of  typhoid  fever  are 
numerous  and  form  a  very  striking  picture. 

Chemotaxis. — Motile    bacteria    possess    the   property    of 
being  attracted  by  certain  substances  (positive  chemotaxis) 


MORPHOLOGY   AND    PHYSIOLOGY   OF    BACTERIA,         125 

and  of  being  repelled  by  others  (negative  chemotaxis). 
Similar  properties  are  widely  distributed  among  living 
cells,  both  animal  and  vegetable. 

CONDITIONS  FAVORABLE  FOR  THE  GROWTH  OF 
BACTERIA. 

Warmth. — Among  the  different  kinds  of  bacteria  forms 
are  said  to  exist  which  multiply  at  temperatures  as  low  as 
o°  C,  while  there  are  species  that  multiply  at  70°  C.  Bac- 
teria which  flourish  at  a  very  high  temperature  (maximum 
about  70°  C.)  are  called  thermophilic.  The  pathogenic 
bacteria  usually  flourish  better  at  a  point  somewhere  near 
the  temperature  of  the  human  body.  This  is  not  necessa- 
rily  the  case  with  the  non-pathogenic  species.  Ordinary 
water  bacteria  thrive  better  at  ordinary  temperatures. 

Sternberg's  method  for  determining  the  thermal  death- 
point  of  a  species  of  bacteria  is  to  draw  portions  of  a  pure 
culture  of  the  organism  into  capillary  tubes  with  expanded 
ends,  when  the  tubes  are  sealed  in  the  flame.  The  tubes 
are  supported  upon  a  glass  plate  placed  in  a  water-bath, 
whose  temperature  is  indicated  by  a  thermometer,  while  a 
uniform  temperature  is  secured  by  stirring.  The  time  of 
exposure  is,  as  a  rule,  ten  minutes.  The  tubes  should  be 
removed  quickly  to  cold  water.  Their  contents  should 
afterwards  be  inoculated  into  bouillon  to  determine  whether 
or  not  the  organisms  have  been  killed. 

Moisture  is  indispensable  to  the  growth  of  bacteria,  and 
drying  causes  the  death  of  certain  kinds,  as,  for  instance, 
the  spirillum  of  cholera. 

Food. — There  are  a  few  species  of  bacteria  that  contain 
chlorophyll,  but  it  is  wanting  in  most  forms.  On  account 
of  the  absence  of  chlorophyll,  bacteria  require,  as  part  of 
their  food,  organic  compounds  containing  carbon,  such  as 
sugar.  They  are  unable,  with  possibly  a  very  few  excep- 


126  MANUAL    OF    BACTERIOLOGY. 

tions,  like  the  nitrifying  bacteria,  to  derive  their  carbon 
from  the  carbon  dioxide  of  the  atmosphere,  or  from  inor- 
ganic carbon  compounds.  Although  some  species  are 
able  to  obtain  nitrogen  from  inorganic  salts,  most  bacteria 
flourish  best  if  organic  substances  containing  nitrogen,  like 
peptone  and  albumen,  are  furnished  them  as  part  of  their 
food.  The  complicated,  unstable,  organic  molecules  with 
high  potential  energy  are  converted  by  them  into  simple 
and  more  stable  compounds  like  carbon  dioxide,  ammonia 
and  water,  with  the  liberation  of  energy.  These  facts  be- 
come manifest  in  connection  with  their  important  work  in 
decomposition,  putrefaction  and  fermentation.  A  culture- 
medium  having  a  slightly  alkaline  or  neutral  reaction  is 
favorable  to  most  bacteria. 

The  prolonged  artificial  cultivation  of  bacteria  may  or 
may  not  modify  their  properties.  The  pathogenic  bacteria 
are  likely  to  undergo  considerable  modification  both  in  the 
quality  and  luxuriance  of  their  growth  and  the  intensity  of 
their  pathogenic  characters. 

The  growth  of  bacteria  may  eventually  be  hindered  by 
the  accumulation  of  the  products  of  their  own  metabolism. 
Many  bacteria  refuse  to  grow  on  culture-media  at  all.  Some 
species  are  extremely  fastidious,  and  can  only  be  propagated 
on  particular  sorts  of  nutrient  substances. 

Relation  to  Oxygen. — Oxygen  is  indispensable  to  the 
growth  of  some  bacteria,  aerobes.  Its  absence  is  equally 
indispensable  to  certain  others,  anaerobes.  Others  still  are 
able  to  flourish  either  in  the  presence  or  absence  of  oxygen, 
facultative  aerobes  or  anaerobes.  The  first-named  varieties 
are  sometimes  called  strict,  or  obligate  aerobes  or  anaerobes. 

Effects  of  Sunlight. — Direct  sunlight  kills  the-  vegeta- 
tive forms  of  bacteria  more  or  less  rapidly,  and  constitutes 
one  of  the  most  efficient  among  the  natural  methods  of  dis- 
infection. Diffuse  daylight  acts  much  more  slowly.  Elec- 


MORPHOLOGY    AND    PHYSIOLOGY    OF    BACTERIA.         127 

trie  light  acts  like  sunlight  or  daylight,  the  results  being  de- 
pendent on  the  intensity  of  the  light.  The  violet  part  of  the 
spectrum  is  most  active. 

The  influence  of  electricity  upon  bacteria  has  not  yet  been 
fully  studied.  Apparently  the  destruction  of  bacteria  re- 
ported as  having  been  effected  by  electricity  was  the  result 
of  electrolysis  of  the  medium. 

It  appears  probable  that  X-rays  do  not  produce  impor- 
tant effects  on  bacteria,  although  further  investigation  of 
this  subject  is  needed.  The  success  which  has  attended  the 
use  of  light  rays  and  X-rays  in  the  treatment  of  lupus  and 
other  diseases  is  not  necessarily  to  be  explained  as  the  result 
of  bactericidal  action  of  the  rays. 


128  MANUAL  OF   BACTERIOLOGY. 


CHAPTER    II. 

PRODUCTS  OF  THE  GROWTH  OF  BACTERIA. 

Phosphorescence. — Bacteria  whose  cultures  exhibit  phos- 
phorescence have  been  found  in  the  ocean  and  in  fish. 

Chromogenic  Bacteria. — Many  bacterial  growths  display 
brilliant  coloring.  The  different  species  of  sarcinse  are 
remarkable  for  forming  highly-colored  growths;  some  of 
them  are  rose-red,  some  orange-yellow,  some  lemon-yellow, 
and  so  on.  The  bacillus  prodigiosus  presents  a  brilliant  red 
growth  whose  rapid  development  is  said  to  have  formed  the 
basis  for  the  so-called  "  Miracle  of  the  Bleeding  Host  "  (see 
page  15).  The  bacillus  pyocyaneus  in  culture  gives  a  bril- 
liant green  fluorescence  and  is  responsible  for  the  color  of 
blue  or  green  pus. 

Bacilli  which  exhibit  a  green  fluorescence  in  cultures  are 
common  in  water.  In  cultures  on  potato  or  agar  the  colors 
of  the  chromogenic  forms  are  usually  well  shown. 

Ferments  or  Enzymes.1 — Many  bacteria  form  ferments 
which  have  the  power  of  dissolving  proteid  substances  in 
a  manner  similar  to  trypsin.  The  liquefaction  of  gelatin 
is  a  familiar  example  of  this  process.  The  property  of 
liquefying  gelatin,  or  otherwise,  is  used  in  classifying  bac- 
teria and  in  determining  the  nature  of  unknown  species. 

Some  bacteria,  as  the  bacillus  coli  communis,  form  fer- 
ments which  act  like  rennet  in  coagulating  milk.  Other 
bacteria  are  capable  of  forming  sugar  from  starch.  Others 
have  the  power  of  changing  cane-sugar  into  glucose. 

1  Consult  Buxton,  "  Mycotic  Enzymes,"  American  Medicine,  July  25, 
1903- 


PRODUCTS    OF   THE    GROWTH    OF    BACTERIA.  129 

Bacteria  which  are  able  to  decompose  cellulose  are  found 
in  the  stomachs  of  ruminant  animals.  Although  it  is  doubt- 
ful whether  the  products  of  cellulose  decomposition  have 
any  nutritive  value,  the  process  is  useful  in  effecting  a  sub- 
division of  the  coarse  food,  consisting  of  grass,  hay,  and 
the  like. 

Some  bacteria  have  the  power  of  decomposing  neutral 
fats  into  fatty  acids  and  glycerin,  after  the  manner  of  the 
fat-splitting  ferment  of  the  pancreatic  juice. 

The  end-products  which  result  from  the  growth  of  bac- 
teria upon  albuminous  nutrient  media  are  very  numerous. 
They  are  complicated  and  not  well  understood.  Among 
these  end-products  may  be  mentioned  peptone,  indol,  skatol, 
phenol,  leucin  and  tyrosin.  Nearly  related  are  the  toxins, 
which  play  an  important  part  in  the  production  of  disease 
by  pathogenic  bacteria.  In  the  decomposition  of  urine  by 
bacteria  the  urea  is  converted  into  ammonium  carbonate. 

The  formation  of  indol  in  cultures  is  an  important  pecu- 
liarity of  certain  bacteria,  which  may  be  tested  as  follows : 
The  bacteria  are  cultivated  in  Dunham's  peptone  solution  or 
in  dextrose-free  bouillon;  after  twenty-four  to  forty-eight 
hours  the  test  may  be  made.  Add  ten  drops  of  concentrated 
sulphuric  acid;  the  development  of  a  rose-color  indicates 
the  presence  of  both  indol  and  nitrites.  If  no  rose-color 
forms,  to  another  tube  add,  first  i  c.c.  of  a  o.oi  per  cent, 
solution  of  sodium  nitrite,  and  then  the  sulphuric  acid.  The 
development  of  a  rose-color  indicates  the  formation  of  indol 
but  not  of  nitrites.  If  there  is  no  rose-color,  no  indol  has 
been  formed.  The  color  appears  usually  in  a  few  minutes, 
but  it  may  only  develop  after  a  somewhat  longer  time.  Con- 
trol tests  must  be  made  upon  tubes  of  the  same  peptone 
solution  but  which  have  not  been  inoculated.  The  reaction 
may  be  hastened  by  warming  slightly.  The  value  of  this 
reaction  will  be  understood  when,  to  give  one  illustration. 


130  MANUAL    OF    BACTERIOLOGY. 

it  is  remembered  that  the  bacillus  coli  communis  produces 
indol  and  the  bacillus  of  typhoid  fever  usually  does  not. 
The  reaction  depends  upon  the  liberation  of  nitrous  acid, 
which,  with  indol,  forms  a  red  color. 

The  change  of  organic  substances  into  more  stable  ones 
does  not  cease  with  the  compounds  mentioned  above.  Cer- 
tain bacteria  of  the  soil  which  will  be  discussed  further  on 
are  able  to  complete  the  conversion  of  ammonia  into  nitrous 
acid  (leading  to  the  formation  of  nitrites)  ;  and  others  still 
that  of  nitrites  into  nitric  acid,  which  at  once  forms  nitrates, 

Formation  of  Acids. — In  the  course  of  their  growth 
many  bacteria  produce  acids,  especially  from  substances 
containing  sugar.  The  power  of  developing  lactic  acid  is 
possessed  by  a  large  number  of  species.  Acetic  acid  is 
another  common  by-product.  Besides  these,  butyric  acid, 
formic  acid,  propionic  acid  and  many  more  are  formed  by 
different  bacteria. 

Development  of  Gas. — The  evolution  of  gas  from  bac- 
terial growths  is  of  frequent  occurrence.  Carbon  dioxide, 
hydrogen  sulphide  and  nitrogen  are  among  the  better 
known  gases  that  may  be  formed.  The  odors  that  arise 
from  cultures  and  that  are  so  characteristic  of  putrefactive 
processes  depend  upon  the  development  of  gases,  or  a  mix- 
ture of  gases,  of  considerable  complexity.  The  bacillus 
aerogenes  capsulatus  leads  sometimes  to  the  formation  of 
gas  in  the  organs  of  the  human  cadaver  within  a  short  time 
after  death. 

T.  Smith  considers  the  formation  of  gases  in  media  con- 
taining sugar  of  importance  in  discriminating  between  dif- 
ferent species.  Bouillon  containing  i  per  cent,  of  dextrose 
(or  lactose,  etc.)  is  the  culture-medium  advised.  The 
test  is  best  conducted  in  a  U-shaped  tube,  closed  at  one  end, 
and  at  the  other  end  provided  with  a  bulb  (Fig.  46).  The 
tube  is  stoppered  with  cotton,  sterilized  by  dry  heat,  after- 


PRODUCTS    OF    THE    GROWTH    OF    BACTERIA.  13! 

ward  filled  with  the  bouillon,  and  sterilized  by  steam  in  the 
usual  manner.  After  the  last  sterilization  it  should  be  tilted 
until  the  closed  end  is  completely  filled  with  the  medium. 
After  it  has  been  inoculated  with  the  species  under  con- 
sideration, any  development  of  gas  will  be  indicated  by  the 
collection  of  the  gas  at  the  closed  end.  The  amount  of  gas 
formed  may  be  estimated  and  its  FlG 

quality  tested.  To  accomplish  the 
latter  fill  the  bulb  with  2  per  cent, 
solution  of  sodium  hydroxide, 
close  the  outlet,  and  tilt  the  tube 
to  allow  the  mixture  to  come  in 
contact  with  the  gas.  After 
shaking,  this  causes  the  absorp- 
tion of  the  carbon  dioxide  and 
diminution  in  the  quantity  of 
gas.  The  portions  which  remain 
may  be  mixed  with  air  and 
ignited,  when  the  presence  of 
hydrogen  and  some  of  its  com- 
pounds will  be  indicated  by  an 
explosion.  (See  The  Detection 
of  Bacillus  coli  communis  in 

,T.  Fermentation-tube. 

\Vater,  Part  II.,  Chapter  III.) 

The  development  of  gas  may  readily  be  tested  by  inocu- 
lating the  bacteria  by  a  deep  puncture  into  agar  containing 
i  per  cent,  of  dextrose  or  other  sugars.  The  development 
of  gas  causes  bubbles  to  form  in  the  agar,  often  to  the  extent 
of  splitting  it,  and  sometimes  forcing  out  the  cotton  plug 
(see  Fig.  68). 

The  activities  of  bacteria  which  have  just  been  enumer- 
ated are  fundamental  to  the  phenomena  which  go  by  the 
names  of  fermentation  and  putrefaction.  These  words 
have  been  defined  differently  at  different  times  and  by  dif- 
ferent writers,  but  in  general  both  are  used  as  names  for 

12 


132  MANUAL    OF    BACTERIOLOGY. 

the  breaking  up  of  complex  organic  compounds  by  micro- 
organisms with  the  formation  of  simpler  compounds.  Fer- 
mentation refers  especially  to  the  formation  of  useful  prod- 
ucts like  alcohol.  The  term  putrefaction  is  employed  chiefly 
for  the  breaking  up  of  nitrogenous  compounds  with  the 
development  of  foul-smelling  gases.  The  term  fermentation 
is  also  applied  to  the  decomposition  of  complex  substances 
through  the  influence  of  unorganized  ferments  or  enzymes. 

The  work  of  bacteria  in  fermentation  and  putrefaction  is 
indispensable  to  the  existence  of  the  organic  world  as  we 
find  it.  Green  plants  convert  the  stable  compounds  of  nitro- 
gen, the  carbon  dioxide  of  the  atmosphere,  and  water  into 
the  complex  and  unstable  albumins  and  carbohydrates  which 
serve  as  food  for  animals.  Animals,  on  the  other  hand,  con- 
vert these  unstable  and  complex  compounds  back  into  simpler 
forms.  The  work  of  changing  them  back  into  the  simple 
and  stable  condition,  in  which  they  serve  as  the  food  for 
plants,  is  performed  by  animal  life  in  part  only,  and  its  com- 
pletion is  left  to  the  activities  of  bacteria.  It  is  the  work  of 
bacteria  in  this  direction  which  we  call  fermentation  and 
putrefaction.  Without  that  work,  as  we  understand  it,  the 
existence  of  life  upon  the  earth  would  soon  come  to  an  end, 
and  the  dead  and  undecomposed  bodies  of  living  things  and 
their  products  of  all  kinds  would  lie  about  unchanged,  as 
they  had  fallen. 

Bacterium  tcnno  is  the  name  formerly  given  to  a  sup- 
posed species  of  bacteria  which  was  credited  with  being 
the  producer  of  putrefaction.  The  individuals  were  rep- 
resented as  being  short  rods,  mostly  going  in  pairs,  and 
actively  motile.  The  term  has  been  abandoned  since  it 
appears  to  have  included  a  number  of  different  species. 


DISTRIBUTION    OF    BACTERIA.  133 


CHAPTER  III. 

DISTRIBUTION  OF  BACTERIA. 

The  Bacteria  of  the  Soil. — Bacteria  are  present  in  the 
soil  in  enormous  numbers — 100,000  or  more  in  i  c.c.  of 
virgin  soil,  according  to  Fliigge.  The  depths  to  which 
they  penetrate  will  depend  upon  the  character  of  the  soil 
and  the  character  of  the  life  upon  it,  and  whether  or  not  it 
has  been  artificially  disturbed,  as  by  cultivation.  In  gen- 
eral, at  a  depth  of  1.25  meters  (about  four  feet)  the  num- 
ber will  have  become  very  small,  and  a  little  deeper  the  soil 
will  be  entirely  sterile. 

The  bacilli  of  tetanus  and  malignant  edema,  and  bacillus 
aerogenes  capsulatus  are  present  in  the  soil  of  many  locali- 
ties. According  to  Woodhead,  certain  savage  tribes  of 
Africa  and  the  East  Indies  use  as  an  arrow-poison  soil  that 
is  capable  of  producing  tetanus.  The  bacillus  of  anthrax 
may  be  found  in  soil  which  has  been  infected  with  this 
organism. 

Most  of  the  bacteria  of  the  soil  are  harmless  or  useful 
saprophytes.1  The  nitrifying  bacteria  described  by  Wino- 
gradsky  and  by  Jordan  and  Richards  belong  to  the  latter 
class.  There  occur  in  soil  organisms  which  have  the  power 
of  converting  ammonia  into  nitrous  acid  which  forms 
nitrites,  and  others  which  complete  the  change  of  nitrites 
into  nitrates.  Both  varieties  are  widely  distributed.  These 
organisms  will  not  grow  on  ordinary  culture-media,  and 
their  cultivation  presents  great  difficulties.  Probably  a  good 
many  bacteria  have  similar  properties  to  some  extent.  The 

1  See   Conn,   "  Agricultural   Bacteriology." 


134  MANUAL    OF    BACTERIOLOGY. 

work  done  by  nitrifying  bacteria  in  making  nitrates  from 
sewage,  manure  and  the  like  is  indispensable  to  most  plant 
life.  Bacteria  have  also  been  credited  with  the  assimilation 
of  free,  atmospheric  nitrogen,  resulting  in  the  addition  of 
a  valuable  proportion  of  nitrogen  compounds  to  the  soil. 
This  is  spoken  of  as  nitrogen  -fixation.  Inasmuch  as  a  large 
part  of  the  excrementitious  products  of  animals  containing 
nitrogen  are  not  retained  in  the  soil,  where  they  may  be 
employed  as  food  by  plants,  but  are  washed  directly  or  in- 
directly into  the  sea  by  means  of  sewage  and  the  rivers,  it 
will  be  seen  that  the  supply  of  nitrogen  compounds  might 
be  in  a  way  to  suffer  gradual  exhaustion.  Furthermore,  it 
has  already  been  noticed  (page  130)  that  one  of  the  products 
of  decomposition  by  bacteria  is  nitrogen,  which  is  not  avail- 
able to  animals  and  most  plants  as  food.  These  facts  have 
met  with  practical  recognition  by  agriculturists  in  the  adop- 
tion of  various  methods  of  fertilizing  the  soil.  It  appears 
that  the  roots  of  peas,  beans,  clover,  alfalfa  and  some  other 
plants  frequently  present  minute  tubercles.  These  tubercles 
are  pathological  growths,  caused  by  the  development  of 
microorganisms  related  to  the  bacteria.  The  organisms 
appear  to  have  the  power  of  assimilating  atmospheric  nitro- 
gen and  of  converting  it  into  nitrogen  compounds.  Experi- 
ments show  that  these  observations  may  be  destined  to  be  of 
great  value  to  the  farmer.1 

The  bacteria  of  the  soil  may  easily  be  studied  in  plate- 
cultures  made  from  small  portions  of  soil  collected  with  the 
necessary  precautions  to  avoid  contamination,  or  plate-cul- 
tures may  be  made  from  sterilized  water  with  which  a  por- 
tion of  the  soil  has  been  properly  mixed.  Anaerobic  bac- 
teria must  be  cultivated  by  the  special  methods  adapted  to 
them. 

1  For  simple  experiments  to  illustrate  these  phenomena  see  Buxton, 
Journal  of  Applied  Microscopy,  September,  1902. 


DISTRIBUTION    OF    BACTERIA.  135 

Bacteria  of  the  Air.— The  bacteria  of  the  air  will  be 
found  for  the  most  part  clinging  to  solid  particles  in  suspen- 
sion in  the  shape  of  dust.  As  has  already  been  stated, 
bacteria  will  not  rise  from  moist  surfaces  unless  forcibly 
removed,  as  by  agitation  or  currents  of  air.  Conditions  of 
dryness  and  wind  tend  to  increase  the  number  of  micro- 
organisms in  the  air.  They  are  fewer  after  a  fall  of  rain 
or  snow,  and  the  number  is  smaller  in  winter  than  in  sum- 
mer. The  air  of  cities  contains  more  germs  than  that  of 
the  country.  The  atmosphere  over  the  sea  and  at  the  tops 
of  high  mountains  is  nearly  or  wholly  free  from  germs. 
The  bacteria  which  do  occur  in  the  air  will  seldom  be 
pathogenic.  Their  character  will  depend  upon  the  character 
of  the  dust.  It  is  obvious  that  dust  which  consists  in  part 
of  the  dried,  pulverized  expectoration  of  cases  of  pulmonary 
tuberculosis  may  contain  tubercle  bacilli.  Anthrax  of  the 
lungs  sometimes  arises  in  men  who  handle  the  wool  of 
sheep  that  were  infected  with  anthrax  (Wool-sorter's  dis- 
ease) ,  and  is  due  to  the  inhalation  of  anthrax  spores  attached 
to  the  wool.  It  is  likely  that  the  atmosphere  in  the  imme- 
diate vicinity  of  cases  of  the  exanthematous  fevers  may 
contain  the  organisms,  whatever  they  may  be,  that  cause 
these  diseases. 

In  a  rough  way  one  may  obtain  some  knowledge  of  the 
character  of  the  organisms  in  the  air  of  a  given  locality  by 
removing  the  cover  of  a  Petri  dish  containing  sterilized 
gelatin  or  agar  for  a  few  minutes,  replacing  it,  and  allow- 
ing the  organisms  to  develop.  In  most  cases  a  large  pro- 
portion of  the  growths  that  appear  will  be  moulds.  Yeasts 
are  also  common,  and  among  the  bacteria  the  micrococci 
are  abundant.  Chromogenic  varieties  are  likely  to  be 
present. 

A  few  studies  of  this  character  will  show  that  the  num- 
ber of  organisms  that  are  present  depends  chiefly  upon 


136  MANUAL   OF    BACTERIOLOGY. 

whether  the  air  is  quiet  or  has  recently  been  disturbed  by 
draughts,  gusts  of  wind,  or  sweeping.  These  facts  are  of 
fundamental  importance  in  laboratory  work,  where  plate- 
cultures  are  being  studied,  if  we  wish  to  avoid  contamina- 
tion of  the  plates.  Among  various  devices  that  have  been 
proposed  for  the  accurate  study  of  the  organisms  of  the 
air,  the  Sedgwick-Tucker  aerobioscope  is  the  simplest  and 
most  accurate.  It  consists  of  a  glass  tube,  one  end  of 
which  is  drawn  out  so  as  to  be  smaller  than  the  other. 
The  small  end  contains  a  quantity  of  fine  granulated  sugar ; 
both  ends  are  plugged  with  cotton,  and  the  instrument  is 

FIG.  47- 


Sedgwick-Tucker  aerobioscope. 

sterilized.  A  definite  quantity  of  air  is  to  be  aspirated 
through  the  large  end,  after  removing  the  cotton,  which 
may  be  done  by  means  of  a  suction-pump  applied  to  the 
other  end,  or  by  siphoning  water  out  of  a  bottle  the  upper 
part  of  which  is  connected  with  the  end  of  the  aerobioscope 
by  means  of  a  rubber  tube.  The  sugar  acts  as  a  filter  and 
sifts  out  of  the  air  the  microorganisms  which  are  contained 
in  it.  Liquefied  gelatin  or  agar  may  be  introduced  into 
the  large  end  of  the  instrument  by  means  of  a  bent  funnel ; 
and,  after  replacing  the  cotton,  it  may  mix  with  the  sugar 
which  dissolves.  The  culture-medium  may  be  spread 
around  the  inside  of  the  larger  portion  of  the  tube  after  the 
manner  of  an  Esmarch  roll-tube.  The  bacteria  which 
were  filtered  out  by  the  sugar  will  develop  as  so  many 
colonies  upon  the  solidified  medium. 

Bacteria  of  Water  and  of  Ice. — The    water    of    rivers, 
lakes  and  the  ocean  always  contains  bacteria.     The  num- 


DISTRIBUTION    OF    BACTERIA.  137 

her  of  organisms  varies  greatly  in  different  places  and 
under  different  conditions.  The  number  of  different  spe- 
cies found  in  water  is  also  very  large.  Ground-water1 
contains  few  or  no  bacteria  under  normal  conditions,  and 
is  therefore  suitable  for  a  source  of  water-supply,  when  a 
sufficient  amount  is  available.  The  possibility  of  contami- 
nation of  the  ground- water  from  unusual  or  abnormal  con- 
ditions should  always  be  eliminated  before  it  is  taken  for 
drinking-water.  Numerous  epidemics  of  typhoid  fever  have 
been  traced  to  contamination  of  wells.  The  location  of  wells 
with  reference  to  privy-vaults  and  other  possible  sources  of 
contamination  should  be  chosen  with  the  greatest  care. 

The  ordinary  bacteria  of  water  are  harmless,  as  far  as 
is  known.2  Bad  odors  and  tastes  in  drinking  water  that  is 
not  polluted  with  putrid  material  are  usually  due  to  minute 
green  plants  (algae).3  The  diseases  most  commonly  dis- 
seminated by  water  are  typhoid  fever  and  Asiatic  cholera, 
and  probably  also  dysentery.  The  spirillum  of  cholera  will 
usually  die  in  natural  water  (not  sterilized  water)  inside 
of  two  or  three  weeks ;  the  bacillus  of  typhoid  fever  will  usu- 
ally die  in  two  or  three  weeks.  Under  exceptional  circum- 
stances these  organisms  may  perhaps  maintain  their  vitality 
for  a  longer  period.  They  appear,  however,  to  be  less 
hardy  than  the  ordinary  water  bacteria.  As  wre  now  un- 
derstand these  diseases,  the  organisms  causing  them  will 
be  present  only  in  a  water-supply  which  has  been  contami- 
nated by  the  excreta  from  a  case  of  the  disease.  Notwith- 
standing the  rapid  death  of  these  organisms  in  water,  they 

1  Ground-water  is  the  water  which — originally  derived   from  rain  or 
snow — sinks  through  superficial  porous  strata,  like  gravel,  and  collects 
on  some  underlying,  impervious  bed  of  clay  or  rock. 

2  See   Fuller  and   Johnson,   "  The   Classification   of   Water   Bacteria," 
Journal  of  Experimental  Medicine,  Vol.   IV.,   p.   609. 

1 "  Contamination  of  Water  Supplies  by  Algae."  G.  T.  Moore  in 
Yearbook  U.  S.  Department  of  Agriculture,  1902. 


138  MANUAL    OF    BACTERIOLOGY. 

may  exist  long  enough  to  infect  individuals  habitually 
drinking  the  water.  Many  epidemics  of  cholera  and  typhoid 
fever  have  been  traced  to  water  polluted  with  the  discharges 
from  cases  of  these  diseases. 

By  self-purification  of  water  is  meant  the  removal  through 
natural  processes  of  contaminating  organisms  such  as  might 
occur  from  the  discharge  of  sewage  into  it.  It  depends 
upon  the  sedimentation  of  the  contaminating  material,  in 
the  form  of  mud,  upon  the  growth  of  the  ordinary  water- 
plants  and  protozoa,  upon  the  exhaustion  of  the  food 
supply  by  the  growth  of  bacteria  themselves,  upon  the 
destructive  influence  of  direct  sunlight,  and  the  dilution  of 
the  matter  added  with  a  large  volume  of  water.1  It  is  not 
usually  to  be  relied  upon  as  a  means  of  freeing  the  water- 
supply  from  pathogenic  bacteria. 

Storage  of  water.  AYhen  water  is  kept  in  large  reser- 
voirs, the  solid  particles  in  it,  including  bacteria,  tend  to  fall 
to  the  bottom.  The  number  of  bacteria  in  a  water-supply 
may  be  considerably  reduced  in  this  way. 

Filtration. — Filtration  on  a  large  scale  has  been  more 
commonly  in  use  in  the  cities  of  Europe  than  elsewhere,  until 
lately.  Similar  filtration-plants  now  exist  in  several  cities 
of  the  United  States. 

Slow  Sand  Filtration. — The  filter  consists  of  successive 
layers  of  stones,  coarse  and  fine  gravel.  The  uppermost 
layers  are  of  fine  sand.  The  whole  filter  is  from  I  to  2 
meters  thick.  The  sand  should  be  60  cm.  in  thickness.  The 
upper  layers  may  be  removed  from  time  to  time,  the  re- 
mainder not  becoming  less  than  30  cm.  in  thickness.  The 
first  water  coming  from  the  filter  is  discarded.  The  actual 
filtration  is  done  largely  by  the  slimy  sediment  which  collects 
on  the  surface  of  the  layer  of  fine  sand.  The  filter-beds  may 
be  several  acres  in  extent,  and  are  protected  by  arches  of 
brick  or  stone.  They  require  renewal  occasionally.  This 

1  See  Jordan,  Journal  of  Experimental  Medicine,  Vol.  V.,  p.  271. 


DISTRIBUTION    OF    BACTERIA.  139 

kind  of  filtration  has  come  largely  into  use  since  the  cholera 
epidemic  of  1892-93,  and  it  appears  to  be  very  effective. 

Mechanical  Filtration. — This  method  of  filtration  is  also 
called  the  American  system.  It  is  more  rapid  than  the  pre- 
ceding method  and  does  not  require  a  large  area  for  filter 
beds.  Although  sand  is  required  also,  filtration  is  accom- 
plished by  a  jelly-like  layer  of  aluminum  hydroxide.  This 
product  is  formed  by  adding  to  the  water  a  small  quantity 
of  alum.  The  carbonates  in  the  water  decompose  the  alum 
and  produce  aluminum  hydroxide.  It  precipitates  as  a 
white,  flocculent  deposit,  entangling  solid  particles,  includ- 
ing bacteria,  as  coffee  is  cleared  with  white  of  egg.  Only  a 
trace  of  alum  should  appear  in  the  water.  This  method  of 
filtration  has  not  been  tested  so  extensively  as  slow  sand 
filtration,  but  seems  likely  to  prove  efficient.  With  water 
poor  in  carbonates,  these  may  have  to  be  added. 

Various  methods  for  the  purification  of  water  by  means 
of  chemicals  have  been  proposed.  The  use  of  ozone  for  this 
purpose  has  met  with  considerable  favor.1 

The  filtration  of  water  on  a  small  scale,  as  is  ordinarily 
done  for  domestic  purposes,  is  generally  entirely  useless. 
The  so-called  Pasteur  filter  of  unglazed  porcelain  is  effec- 
tive if  it  is  properly  constructed  and  if  the  filter-tubes  are 
sterilized  by  heat  frequently  (every  few  days) — conditions 
which  are  seldom  complied  with.  Distillation  of  water,  or 
thorough  boiling  will  usually  be  the  most  practical  method 
for  sterilizing  drinking-water. 

Collection  of  Samples. — Samples  from  the  water-supply 
of  a  city  may  be  drawn  from  the  faucet,  but  the  water 
should  first  be  allowed  to  run  for  half  an  hour  or  longer. 
From  other  sources  the  supply  should  be  collected  in  ster- 
ilized tubes  or  bottles,  taking  care  to  avoid  contamination. 
Sternberg  bulbs  (see  Fig.  38)  will  be  found  useful  for 

1  Consult    "  Disinfection    and    Disinfectants/'    Rosenau,    1902. 


J4O  MANUAL    OF    BACTERIOLOGY. 

small  samples.  These  samples  should  be  examined  as 
quickly  as  possible,  for  the  water  bacteria  increase  rapidly 
in  number  after  the  samples  have  been  collected.  When 
transportation  to  some  distance  is  unavoidable  the  samples 
should  be  packed  in  ice. 

The  number  of  bacteria  may  be  determined  by  making' 
plates  of  a  definite  quantity  of  the  water  with  gelatin  or 
agar.  The  amount  examined  ordinarily  is  I  c.c.  When 
the  number  of  bacteria  is  very  large,  a  smaller  quantity 
must  be  taken,  and  it  may  be  necessary  to  dilute  the  sample 
ten  times  or  more  with  sterilized  water.  The  amount  should 
be  measured  with  a  sterilized,  graduated  pipette.  The  water 
is  to  be  mixed  with  liquefied  gelatin  or  agar  in  a  tube  which 
has  been  allowed  to  cool  after  melting.  After  thorough 
mixing,  remove  the  plug,  burn  the  edge  of  the  tube  in  the 
flame,  hold  in  a  nearly  horizontal  position  until  cool,  and 
pour  into  a  sterilized  Petri  dish.  The  number  of  colonies 
may  be  counted  on  the  third  or  fourth  day;  the  later  the 
better,  as  some  forms  develop  slowly  and  may  not  present 
visible  colonies  for  several  days;  but  the  plates  are  often 
spoiled  after  three  or  four  days  by  the  profuse  surface 
growths  of  certain  forms  or  by  the  rapid  liquefaction  of 
gelatin,  if  that  be  used,  by  other  forms.  The  number  of 
colonies  that  develop  is  supposed  to  represent  the  number 
of  individual  bacteria  contained  in  the  quantity  measured. 
That  will  probably  not  always  be  the  case,  however,  as 
colonies  may  develop  from  a  clump  of  bacteria  which  have 
not  been  separated  from  one  another  by  the  mixing  process. 
Abbott  has  shown  that  the  number  of  colonies  is  usually 
larger  on  gelatin  plates  than  upon  agar  plates,  and  at  the 
room  temperature  than  in  the  incubator.  This  observation 
illustrates  the  fact  that  there  are  doubtless  many  kinds  of 
bacteria  that  do  not  find  favorable  conditions  for  develop- 
ment on  ordinary  culture-media.  The  reaction  of  the 


DISTRIBUTION    OF    BACTERIA.  I^-T 

medium  has  an  important  influence  upon  the  development  of 
these  water  bacteria  in  plate-cultures. 

When  the  number  of  colonies  is  small,  there  is  no  diffi- 
culty in  counting  them  as  they  appear  in  the  ordinary 
Petri  dish.  When  the  number  is  large  some  kind  of  me- 
chanical device  may  be  used  to  assist  counting.  The 
Wolffhiigel  plate  is  a  large  square  of  glass  resting  in  a 
wooden  frame  painted  black.  The  glass  plate  is  ruled 
in  squares.  It  was  designed  particularly  with  reference 
to  the  form  of  plate-cultures  first  made  by  Koch.  The 
Petri  dish,  however,  may  be  placed  upon  the  glass  plate 
and  the  cross  lines  be  used  to  assist  in  counting.  Lafar, 
Pakes  and  Jeffer  recommend  a  surface  painted  black,  ruled 
with  white  lines  which  represent  the  radii  of  a  circle,  which 
may  be  still  further  subdivided  by  other  lines.  Many  find 
counting  easier  when  a  black  surface  divided  into  squares 
is  employed.  An  ordinary  card  with  a  smooth  black  sur- 
face divided  into  squares  by  white  lines  may  be  placed  under 
a  Petri  dish  and  will  be  found  to  serve  very  well.  For  the 
mere  examination  of  the  colonies  no  better  surface  can  be 
devised  than  the  ferrotype  plate  used  by  photographers. 
The  examination  of  the  colonies  will  be  easier  if  a  small 
hand-lens  be  used.  Care  must  be  taken  not  to  mistake  air- 
bubbles  or  particles  of  dirt  for  colonies  of  bacteria. 

In  any  case,  if  possible,  all  the  colonies  in  the  plate 
should  be  counted.  The  number  contained  within  several 
squares  may  be  counted  and  the  average  taken;  knowing 
the  size  of  the  squares  and  the  area  of  the  plate,  the  num- 
ber contained  in  the  whole  plate  may  be  calculated.  Such 
estimations,  however,  are  likely  to  give  results  very  wide 
of  the  truth. 

The  plating  may  be  done  by  rolling  the  medium  after  the 
manner  of  Esmarch.  When  the  number  of  colonies  is  not 
large  this  may  serve  very  well.  Counting  may  be  assisted  by 


142  MANUAL    OF    BACTERIOLOGY. 

drawing  lines  with  ink  on  the  outer  surface  of  the  test-tube. 

It  has  been  said  that  a  water-supply  containing  no  more 
than  500  bacteria  per  cubic  centimeter  is  to  be  regarded  as 
safe,  one  having  between  500  and  1000  is  to  be  looked  upon 
with  suspicion,  and  that  where  there  are  more  than  1000 
to  the  cubic  centimeter  the  water  is  unfit  for  drinking  pur- 
poses. It  is  obvious,  however,  that  the  character  of  the 
bacteria  is  of  prime  importance;  that  pathogenic  organ- 
isms may  occasionally  be  present,  even  when  the  number 
of  bacteria  to  the  cubic  centimeter  is  small.  But  knowing 
the  number  usually  found  in  a  good  water-supply,  any 
sudden  variation  above  that  number  is  to  be  looked  upon 
with  suspicion.  An  increase  is  to  be  expected  when  the 
water  has  been  subjected  to  unusual  agitation  from  \vinds 
or  currents. 

The  detection  of  pathogenic  bacteria  in  water1  involves 
great  difficulties,  and  our  knowledge  in  this  direction  is  very 
meagre.  Koch  and  several  others  have  reported  finding 
the  spirillum  of  Asiatic  cholera  in  water.  The  examination 
of  water-supplies  for  this  organism  has  disclosed  the  fact 
that  bacteria  resembling  the  organism  of  cholera  in  many 
respects  are  not  uncommon  in  water.  This  adds  to  the  diffi- 
culty of  detecting  the  cholera  germ  in  water. 

The  bacillus  of  typhoid  fever  has  many  times  been  de- 
scribed as  occurring  in  water-supplies  suspected  of  being 
contaminated  with  the  excreta  of  cases  of  the  disease.  The 
interpretation  of  these  observations  is  at  present  doubtful. 
It  is  now  known  that  several  forms  of  bacteria  exist  which 
closely  resemble  the  bacillus  of  typhoid  fever,  and  which 
would  make  its  recognition  in  an  unknown  specimen  very 
difficult. 

It  will  at  once  be  appreciated  that  the  number  of  cholera 
and  typhoid  organisms  necessary  to  contaminate  a  consid- 

1  See  also  articles  in  Part  IV.  on  the  bacillus  of  typhoid  fever,  bacillus 
coli  communis  and  spirillum  of  cholera. 


DISTRIBUTION    OF    BACTERIA.  143 

erable  body  of  water,  and  sufficient  to  cause  an  outbreak  of 
the  disease  among  some  of  the  people  drinking  the  water, 
may  still  be  so  small  that  many  different  cubic  centimeters 
of  the  water  might  be  studied  before  a  single  one  of  the 
specific  organisms  would  be  encountered.  Anyone  who 
has  examined  plates  made  from  samples  of  water  will  recog- 
nize the  difficulty  of  detecting  one  or  a  few  colonies  of  the 
bacteria  of  cholera  or  typhoid  fever  among  a  hundred  or 
more  colonies  of  ordinary  water-bacteria.  The  existence  of 
contamination  with  animal  excreta  might,  however,  be  indi- 
cated by  finding  the  bacillus  coli  communis,  whose  detection 
offers  a  greater  prospect  of  success.  It  is  not  certain  just 
how  much  importance  is  to  be  attributed  to  the  presence  of 
small  numbers  of  the  colon  bacillus  in  water.1  Until  our 
knowledge  is  more  complete  any  suspicious  water  should  be 
discarded. 

Certain  devices  have  been  adopted  to  hasten  the  develop- 
ment of  the  desired  bacteria  and  to  retard  that  of  the  ordi- 
nary water-bacteria.  Among  these  may  be  mentioned  the 
influence  of  the  heat  of  the  incubator,  which  will  hasten  the 
growth  of  organisms  derived  from  the  human  body,  and 
which  retards  the  growth  of  water-bacteria.  Another  is 
the  addition  of  a  solution  of  peptone  to  a  large  quantity  of 
the  water  to  be  examined  with  a  view  to  assisting  the 
development  of  the  desired  bacteria  by  furnishing  them 
suitable  food  for  growth.  In  another  method  (Parietti's) 
small  quantities  of  carbolic  acid  are  added  to  bouillon  and 
mixed  with  the  water,  with  a  view  to  retarding  the  develop- 
ment of  all  except  typhoid  and  colon  bacilli.  Suspected  bac- 
teria may  be  tested  by  inoculation  into  animals ;  the  pos- 
session of  pathogenic  properties  creates  a  probability  in 
favor  of  their  having  come  from  some  contamination  with 
animal  excreta. 

Jordan,  Journal  of  Hygiene,  Vol.  I.,  1901.  Savage,  Journal  of  Hy- 
giene, Vol.  II.,  1902.  Winslow  &  Hunnewell,  Journal  Medical  Research, 
Vol.  VIII.,  1902. 


144  MANUAL    OF    BACTERIOLOGY. 

Detection  of  Bacillus  coli  communis  in  Water. — To  each  of  a  number 
of  fermentation-tubes  containing  i  per  cent,  dextrose-bouillon  add  some 
of  the  suspected  water  (.1  to  I  c.c.).  Place  in  the  incubator.  Each  day 
mark  the  amount  of  gas  that  has  formed  in  the  closed  arm.  In  three 
days  B.  coli  communis  should  render  the  bouillon  strongly  acid  and 
produce  50  per  cent,  of  gas  (or  about  that  amount).  From  tubes  show- 
ing these  characters,  plates  may  be  made  and  the  usual  tests  for  the 
colon  bacillus  applied.1  (See  Part  IV.) 

Ice. — The  bacteriological  examination  of  ice  differs  in 
no  respect  from  that  of  water.  Although  development  may 
be  arrested,  the  vitality  of  bacteria  is  not  necessarily  im- 
paired by  freezing.  Prudden  found  the  bacillus  of  typhoid 
fever  alive  in  ice  after  more  than  one  hundred  days.  How- 
ever, Sedgwick  and  Winslow  have  stated  that  when  typhoid 
bacilli  are  frozen  in  water  the  majority  of  them  are  de- 
stroyed. Nevertheless,  it  is  safest  to  have  the  source  from 
which  ice  is  taken  as  carefully  scrutinized  as  that  of  the 
water-supply,  especially  in  view  of  the  universal  habit  of 
cooling  water  in  the  summer  time  by  adding  ice  directly  to 
the  water.  It  is  better  to  cool  water  and  articles  of  food  by 
surrounding  the  vessels  containing  them  with  ice.2 

Bacteria  of  Milk  and  Other  Foods."— Of  the  different 
food  substances,  milk  is  probably  the  most  important  from 
a  bacteriological  point  of  view.  In  the  first  place,  most 
other  foods  are  cooked  before  eating.  Furthermore,  cow's 
milk  constitutes  a  large  part  of  the  food  of  many  young 
infants  who  are  highly  susceptible  to  certain  bacteria,  or  to 
substances  in  the  milk  itself,  after  it  has  undergone  certain 
alterations  due  to  bacteria.  The  milk  of  the  healthy  cow 
as  it-  is  secreted  in  the  mammary  gland  is  sterile;  how- 
ever, after  milking  the  cow  a  little  milk  generally  remains 

1  T.  Smith,  American  Journal  Medical  Sciences,  Vol.  no,  1895. 

3  Clark,  "  Bacterial  Purification  of  Water  by  Freezing,"  Reports  Amer- 
ican Public  Health  Association,  Vol.  XXVII. ,  1901. 

3  See  Conn,  "  Bacteria  in  Milk  and  its  Products,"  1903.  Russell, 
"  Dairy  Bacteriology." 


DISTRIBUTION    OF    BACTERIA.  145 

in  the  milk-ducts  and  in  the  lower  part  of  the  teat  in  which 
numerous  bacteria  will  have  developed  before  the  next 
milking-time.1  The  first  milk  obtained  at  a  milking  should 
therefore  be  discarded,  as  it  may  contain  an  excessive 
number  of  bacteria. 

Contamination  with  bacteria  may  occur  from  the  outer 
surface  of  the  udder  of  the  cow,  the  hands  of  the  milker  or 
dirty  pails,  or  through  agitation  of  the  air  of  the  stable,  and 
in  other  ways  readily  conceived  of.  Bacillus  coli  commu- 
nis  is  often  found  in  milk.  Excluding  the  tubercle  bacillus, 
the  organisms  which  contaminate  milk  will  be  pathogenic 
only  in  exceptional  cases.  Occasionally  typhoid  fever, 
cholera,  and  possibly  scarlet  fever,  diphtheria  and  other 
diseases  are  disseminated  by  means  of  contaminated  milk. 
In  the  case  of  typhoid  fever,  it  is  probable  that  the  milk  cans 
have  been  washed  with  polluted  water;  after  the  cans  were 
filled,  a  few  typhoid  bacilli  left  in  drops  of  water  in  the 
cans,  might  multiply  enormously.  Streptococci  have  been 
found  quite  frequently  in  the  milk  sold  in  cities.2  The 
mixture  with  the  milk  of  non-pathogenic  organisms  from 
the  air,  and  their  growth,  may  induce  changes  in  it  which 
render  it  unfit  for  consumption,  and  even  poisonous.  These 
alterations  may  be  evident  to  the  senses,  as  the  ordinary 
lactic  acid  fermentation  (souring  of  milk),  or  they  may  not. 
The  character  of  the  alterations  doubtless  varies  much  with 
the  temperature  and  with  the  character  of  the  contaminating 
bacteria.  Summer  temperatures  of  course  favor  decom- 
position and  fermentation.  Specialists  in  children's  diseases 
attribute  to  alterations  in  milk  with  the  formation  of  poison- 
ous substances  a  preeminent  influence  in  the  production  of 
the  intestinal  disorders  of  infancy  so  common  in  the  summer. 

1  See    Harrison    and    dimming,    "  The    Bacterial    Flora    of    Freshly 
Drawn  Milk,"  Journal  of  Applied  Microscopy,  November,  1902. 

2  See  Reed  and  Ward,  "  The  Significance  of  the  Presence  of  Strepto- 
cocci in  Market  Milk,"  American  Medicine,  February  14,  1903. 


146  MANUAL    OF    BACTERIOLOGY. 

Poisoning  with  milk,  ice-cream  or  cheese  is  not  rare,  as 
is  well  known.  There  are  many  records  of  whole  compa- 
nies of  individuals  having  been  taken  violently  ill  after 
having  eaten  one  of  these  foods  from  the  same  source  of 
supply.  The  symptoms  in  such  cases  resemble  those  pro- 
duced by  irritant  mineral  poisons  such  as  arsenic :  nausea 
and  vomiting,  vertigo,  dryness  of  the  mouth,  sense  of  burn- 
ing and  constriction  in  the  throat,  difficulty  in  swallowing, 
cramps  and  griping  pain  in  the  bowels,  constipation  or 
diarrhea,  general  prostration  or  even  collapse.  Vaughan 
isolated  from  poisonous  cheese  a  ptomaine  which  he  called 
tyrotoxicon.  It  appears,  however,  that  other  toxins  may 
be  present  in  cheese,  and  that  tyrotoxicon  is  a  somewhat 
rare  poison.  Vaughan  believes  that  bacteria  of  the  colon 
group  play  an  important  part  in  producing  poisons  in  milk 
and  cheese. 

To  prevent  the  alteration  by  bacteria  of  milk  intended 
to  be  the  food  of  infants,  the  practice  of  sterilizing  milk 
has  been  largely  in  vogue.  Unfortunately,  during  steriliza- 
tion the  milk  undergoes  some  kind  of  alteration  which 
makes  it  disagree  with  certain  infants.  Furthermore, 
among  the  organisms  which  would  be  likely  to  contam- 
inate milk  the  bacilli  of  hay  and  potato,  whose  spores  are 
so  excessively  resistant,  would  be  prominent,  and  they  are 
not  killed  by  any  process  to  which  the  milk  intended  for 
an  infant's  consumption  could  possibly  be  subjected  in  the 
household.  Least  of  all  can  sterilization  be  expected  to 
purify  milk  in  which  bacterial  poisons  are  already  formed. 

The  process  called  pasteurization  is  designed,  not  to  ster- 
ilize the  milk  completely,  but  to  destroy  the  vegetative 
forms  of  bacteria,  and  to  destroy  the  ordinary  pathogenic 
bacteria  with  which  the  milk  might  possibly  be  contami- 
nated.1 The  milk  is  subjected  to  a  temperature  of  only 

1  See  Journal  of  Experimental  Medicine,  Vol.  IV.,  p.  217.  "The  Ther- 
mal Death-point  of  Tubercle  Bacilli  in  Milk,"  etc.,  by  T.  Smith. 


DISTRIBUTION    OF    BACTERIA.  147 

about  70°  to  75°  C.  This  temperature  is  less  likely  to  pro- 
duce alteration  in  the  milk  than  sterilization  by  steam  at 
100°  C.  According  to  Freeman,  milk  which  had  been  pas- 
teurized at  75°  C.  and  distributed  among  the  poor  people  of 
New  York  City,  whose  homes  were  not  supplied  with  ice, 
usually  kept  very  well  even  in  the  summer  time  (see  p.  67). 

The  number  of  bacteria  in  milk  may  be  reduced  con- 
siderably by  the  use  of  the  centrifuge  (separator). 

It  has  been  undertaken  recently  to  do  away  as  far  as 
possible  with  the  contamination  usually  inevitable  in  the 
barnyard  and  stable  by  the  use  of  extraordinary  measures 
to  keep  the  cows,  and  especially  their  udders,  clean;  also 
the  hands  of  the  milker  and  the  milk-pails ;  and  by  sprink- 
ling the  floor  of  the  milk-room  to  prevent  dust.1  The  milk 
is  to  be  transported  to  the  city  on  ice.  Milk  which  has 
been  collected  in  this  manner  is  furnished  in  several  cities 
in  the  United  States.  The  cattle  from  which  the  milk  is 
derived  are  regularly  inspected  by  veterinary  surgeons  as 
well  as  subjected  periodically  to  the  tuberculin  test.  The 
surroundings  and  drainage  of  the  stables  are  investigated 
by  physicians  and  sanitary  engineers.  The  milk  is  also 
regularly  analyzed  by  a  chemist.  It  has  been  found  possi- 
ble to  reduce  the  number  of  bacteria  in  milk  very  notice- 
ably. This  milk  is  of  course-  sold  at  a  considerably  higher 
price  than  ordinary  milk. 

The  number  of  bacteria  which  occur  in  samples  of  milk 
varies  greatly.  In  ordinary  milk  as  furnished  by  milkmen 
the  number  of  bacteria  to  the  cubic  centimeter  is  usually 
many  thousands  to  millions;  grocer's  milk  may  contain 
hundreds  of  thousands  to  millions  of  bacteria  to  the  cubic 
centimeter;  frequently  figures  are  reached  which  are  be- 
yond computation. 

Human  milk  often   contains   the   staphylococcus   epicler- 

3  See  W.  H.  Park,  Journal  of  Hygiene,  Vol.  I.,  1901. 
13 


148  MANUAL    OF    BACTERIOLOGY. 

midis  albus,  and  not  seldom  the  staphylococcus  pyogenes 
aureus,  under  normal  conditions. 

Of  the  different  pathogenic  bacteria  liable  to  furnish  a 
source  of  danger  in  milk,  the  most  important  is  the  bacillus 
tuberculosis.  Tuberculosis  is  a  disease  to  which  cattle  are 
exceedingly  prone.  There  is  good  reason  to  believe  that 
infants  acquire  tuberculosis  through  taking  as  food  the  milk 
of  tuberculous  cows,  although  the  danger  from  this  source 
has  probably  been  overestimated.  The  milk  of  tubercu- 
lous cows  may  contain  tubercle  bacilli  when  there  is  no 
tuberculous  disease  of  the  udder.1  The  frequency  of  tuber- 
culosis among  milch  cows  sometimes  becomes  as  high  as  25 
per  cent.,  or  even  more.  Butter  derived  from  the  milk  of 
such  cows  may  contain  tubercle  bacilli.  The  proper  man- 
ner for  the  States  to  deal  with  this  problem,  for  it  is  one 
that  doubtless  will  fall  to  the  individual  States,  has  not  yet 
been  determined.  The  cost  of  killing  such  a  large  number 
of  valuable  cows  would  be  very  great.  Furthermore,  it  is 
by  no  means  certain  that  this  procedure  would  eradicate 
the  disease.  The  flesh  of  cattle  also  is  capable  of  trans- 
mitting tuberculosis,  but  is  a  smaller  source  of  danger  on 
account  of  the  universal  practice  in  the  United  States  of 
thoroughly  cooking  beef. 

"  Ripening  "  of  cream  and  cheese  is  due  to  the  growth  of 
bacteria  which  produce  agreeable  flavors  in  the  butter  and 
cheese.  Molds  are  also  important  in  the  ripening  of  some 
kinds  of  cheese.2 

In  examining  milk  for  bacteria  the  number  may  be  esti- 
mated by  precisely  the  same  technique  as  is  used  for  the 
estimation  of  bacteria  in  water,  except  that  the  milk  must 
be  diluted;  otherwise  the  plates  are  rendered  opaque  by 

1  See   Mohler,   "  Infectiveness  of  Milk  of  Cows   which   have  Reacted 
to  the  Tuber  nlin  Test,"  U.   S.  Dept.   Agriculture,  Bureau  Animal  In- 
dustry, Bull.  No.  44,  1903. 

2  Conn,  "  Agricultural  Bacteriology." 


DISTRIBUTION    OF    BACTERIA.  149 

the  fat.  It  may  be  diluted  one  hundred  times  with  steril- 
ized water;  when  the  number  of  bacteria  is  very  great  a 
second  dilution  may  be  required.  Estimations  based  upon 
such  high  dilutions  can  only  be  approximate.  The  quan- 
tity taken  for  examination  may  be  o.i  to  i  c.c.  Plates 
should  be  made  immediately  upon  collection  of  the  sample. 
If  the  milk  stands  for  a  few  hours  at  the  room  temperature 
in  the  laboratory,  the  number  of  bacteria  will  become 
enormously  increased. 

The  detection  of  a  particular  kind  of  pathogenic  bacterfa 
in  milk  or  butter  involves  very  great  difficulties.  Staining 
of  bacteria  in  milk  may  be  done  by  the  usual  methods,  but 
the  results  are  rendered  unsatisfactory  by  the  oil  in  the  milk. 
The  demonstration  of  tubercle  bacilli  by  staining  methods  is 
likely  to  involve  many  difficulties.  In  this  connection  it  is 
necessary  to  remember  the  group  of  bacilli  which  resemble 
the  tubercle  bacillus  in  resisting  decolor ization  with  acids 
after  staining.  (See  p.  44.)  The  procedure  of  injecting 
milk  into  guinea-pigs  has  been  resorted  to  largely,  but  the 
results  are  only  obtained  after  the  lapse  of  weeks,  when  the 
development  of  tuberculosis  in  the  guinea-pigs  would  indi- 
cate that  the  milk  was  tuberculous,  provided  that  control 
guinea-pigs  remained  healthy.  Furthermore,  the  other  acid- 
proof  bacilli  which  may  occur  in  milk  or  butter  are  capable 
of  producing  nodules  resembling  tubercles.1  (See  Bacillus 
tuberculosis,  Part  IV.)  The  most  satisfactory  plan  will  be 
to  apply  the  tuberculin  test  to  the  cow  from  which  the  milk 
was  derived. 

Among  the  other  articles  of  food,  those  are  to  be  most 
carefullv  scrutinized  which  are  to  be  eaten  after  little  or  no 
cooking,  particularly  salads,  green  vegetables,  fruits,  and 
the  like.  Under  exceptional  circumstances  they  may  be- 
come agents  for  conveying  infectious  diseases.  Conn 

1  Rabinowitsch,  Zeitschrift  f.  Hygiene,  Bd.  XXXVII. ,  p.  439. 


15O  MANUAL    OF    BACTERIOLOGY. 

showed  that  there  was  good  reason  for  attributing  an  epi- 
demic of  typhoid  fever  among  students  at  Middletown, 
Connecticut,  to  raw  oysters.  After  having  been  collected 
from  the  oyster-beds,  these  oysters  were  placed  in  a  small 
stream  to  fatten,  where  they  were  exposed  to  contamination 
from  a  sewer.  Into  this  sewer  the  discharges  of  a  case  of 
typhoid  fever  were  found  to  have  been  running  at  the  time 
when  the  oysters  were  fattening.  An  epidemic  at  Atlantic 
City,  New  Jersey,  in  1902,  was  traced  to  nearly  similar 
causes  and  conditions.1 

The  ordinary  processes  for  curing  and  salting  meat  can- 
not be  relied  upon  to  destroy  pathogenic  bacteria. 

Cases  of  poisoning  by  eating  oysters,  fish,  meat  in  the 
form  of  sausage  or  canned  meat,  and  other  articles  of  food 
are  not  rare.  These  cases  belong  to  the  same  class  as 
those  poisoned  by  milk  and  cheese  already  mentioned.  They 
are  due  to  products  of  bacterial  decomposition.  Such  affec- 
tions are  quite  commonly  called  "  ptomaine  poisoning/'  al- 
though the  poisons  are  not  ptomaines  in  most  cases.  Prob- 
ably a  number  of  bacteria  exist  which  are  capable  of  affecting 
changes  in  meat  and  other  foods  either  before  or  after 
ingestion.  Among  these  are  an  anaerobic  bacillus  described 
by  Van  Ermengem  (B.  botulinus),  bacillus  enteridis  (Gaert- 
ner)  and  members  of  the  groups  of  which  B.  coli  communis 
and  B.  proteus  are  types.2 

1  Philadelphia  Medical  Journal,  November  I,  1902. 

2  See  Vaughan  and  Novy,  "  The  Cellular  Toxins,"  1902.     Ohlraacher, 
"  Food-Intoxication  from  Oatmeal,"  Journal  of  Medical  Research,  Vol. 
VII.,   p.   420.     Galeotti   and    Zardo,    Centralblatt  f.    Bakteriologic,   Vol. 
XX.vL,  1902,  Orig.  p.  593. 


THE    BACTERIA    OF    THE    NORMAL    HUMAN    BODY. 


CHAPTER   IV. 

THE    BACTERIA   OF    THE    NORMAL    HUMAN    BODY. 

THE  numerous  solid  tissues  and  organs  of  the  human 
body,  the  fluids  circulating  in  the  interior  like  the  blood 
and  lymph,  and  the  cavities  that  have  no  connection  with 
the  outer  world,  are  entirely  free  from  bacteria.1  So  also 
the  maxillary,  ethmoidal  and  frontal  sinuses,  middle  ear,2 
urinary  bladder,  uterus  and  Fallopian  tubes,  and  to  a  less 
extent  the  lungs  and  gall-bladder,3  although  having  external 
connections,  are  usually  sterile  when  in  a  healthy  condition. 
When  bacteria  do  enter  the  tissues  from  any  of  the  surfaces 
their  progress  is  checked  by  means  of  the  activities  of  the 
cells  or  fluids  of  the  body,  and  if  they  succeed  in  penetrating 
to  any  considerable  distance  their  advance  is  usually  arrested 
by  the  nearest  group  of  lymph-nodes,  which  appear  to  be 
important  safeguards  for  preventing  the  dissemination  of 
bacteria  throughout  the  body.  As  a  rule,  the  secretions  of 
the  mucous  membranes  are  inimical  to  bacteria. 

The  skin,  as  might  be  expected,  is  liable  to  have  upon 
it  numerous  bacteria,  especially  micrococci,  and  moulds. 

1  This  view  is  not  upheld  by  the  experiments  of   Ford,   who   found 
small  numbers  of  bacteria  in   the  normal   organs   of  rabbits,   cats   and 
dogs    in   the   majority   of   those    examined.      The   species   of   bacteria 
obtained  were  mostly  common  saprophytes,  and  to  some  extent  constant 
in  the  same  kind  of  animal.     Journal  of  Hygiene,  Vol.  I.,  1901. 

2  Calamida  and  Bertarelli,  Centralblatt  f.  Bakteriologie,  Vol.  XXXII., 
1902,  Orig.  p.  428.     Torne,  Ibidem,  XXXIII.,  1903,  p.  250.     Hasslauer, 
Ibidem,  Ref.   XXXII.,  p.    174.     An   examination  of  these  articles   will 
show  that  investigators  disagree  somewhat,  with  regard  to  the  sterility 
of  these  cavities. 

3  See  Review  on  the  "  Bacteriology  of  the  Gall-Bladder  and  its  Ducts," 
American  Journal  Medical  Sciences,  Vol.  123,  p.  372. 


152  MANUAL    OF    BACTERIOLOGY. 

The  staphylococcus  pyogenes  aureus,  the  streptococcus 
pyogenes,  the  bacillus  pyocyaneus  and  the  bacillus  coli 
communis  sometimes  occur  on  the  skin.  According  to 
Welch,  it  always  contains  the  staphylococcus  epidermidis 
albus,  which  may  be  a  form  of  the  staphylococcus  pyogenes 
albus.  This  organism  is  of  some  importance  to  surgeons 
on  account  of  its  relation  to  the  cleansing  of  the  skin  before 
operations.  It  seems  impossible,  by  any  amount  of  clean- 
ing, to  dislodge  all  of  the  germs  in  the  skin,  especially 
those  under  the  nails. 

The  bacteria  of  the  exposed  mucous  membranes  like  the 
conjunctiva  and  the  nasal  cavity1  and  the  mouth  cavity  will 
naturally  be  very  fluctuating  both  in  quantity  and  quality; 
they  will  be,  in  fact,  those  which  happen  to  fall  upon  the 
surface  or  to  be  drawn  in  from  the  external  air. 

In  the  mouth,  however,  there  is  a  certain  group  of  organ- 
isms more  or  less  characteristic  of  it,  many  of  which  have 
not  been  successfully  cultivated.  These  have  been  thor- 
oughly studied  by  Miller,  to  whose  works  students  are  re- 
ferred.2 

Several  species  of  spirilla  have  been  discovered  in  the 
mouth  and  are  found  along  the  margins  of  the  gums.  The 
leptothrix  buccalis,  and  related  organisms  which  have  a 
long,  ribbon-like  form,  also  occur  in  the  mouth.  The  mi- 
crococcus  lanceolatus  (or  pneumococcus)  appears  to  be 
present  in  many  human  mouths.  In  15  to  20  per  cent,  of 
human  mouths  this  organism  is  sufficiently  virulent  to  pro- 
duce fatal  septicemia  when  inoculated  into  susceptible  ani- 
mals. Pyogenic  bacteria,  especially  streptococci,  occur  fre- 
quently, although  not  regularly,  in  the  mouth.  Putrefactive 

1  Hasslauer,  "  Die  Bakterien  flora  der  gesunden  und  kranken  Nasen- 
schleimhaut,"  L'cntralb'.utt  f.  Bakteriologic,  Vol.  XXXIII.,  1902,  Orig. 
p.  47. 

-  Miller,  "  Microorganisms  of  the  Mouth."  For  a  recent  review  on 
the  bacteria  of  the  month,  see  Madzar,  Ccntralblatt  f.  Bakteriologic,  Vol. 
XXXi,  1902,  Ref.  p.  489;  Vol.  XXXII.,  p.  609. 


THE    BACTERIA    OF    THE    NORMAL    HUMAN    BODY.      153 

bacteria  acting  on  particles  of  food  about  the  teeth  produce 
the  bad  odor  from  the  mouths  of  persons  of  careless  habits. 
According  to  Miller,  bacteria  play  an  important  part  in  the 
production  of  dental  caries.  Certain  of  the  bacteria  of  the 
mouth  produce  fermentation  in  the  vicinity  of  the  teeth  with 
the  formation  of  acids,  which  dissolve  the  calcium  salts  of 
the  teeth.  The  softening  and  destruction  of  the  decalci- 
fied matrix  is  then  accomplished  by  other  and  liquefying 
forms. 

The  expired  air  coming  from  the  mouth  and  nose,  con- 
trary to  the  popular  notion,  is  free  from  bacteria,  excepting 
those  which  become  forcibly  detached,  as  by  efforts  of 
sneezing  and  coughing. 

Among  the  other  exposed  mucous  surfaces,  the  urinary 
meatus  and  the  vagina  may  be  included.  The  urinary 
meatus  and  at  least  part  of  the  urethra  will  be  found  to  con- 
tain bacteria,  which,  in  health,  should  be  non-pathogenic, 
although  interest  attaches  to  the  fact  that  diplococci  have 
been  described  which  behaved  with  stains  in  the  same  man- 
ner as  the  gonococcus  (pseudo-gonococci). 

There  has  been  much  dispute  as  to  whether  or  not  the 
pyogenic  bacteria  occur  in  the  vagina  normally.  It  is  prob- 
able that  the  healthy  vagina  is  in  most  cases  free  from  the 
pyogenic  bacteria ;  although  bacteria  of  some  sort  are  always 
present,  and  the  pyogenic  bacteria  may  exceptionally  be 
found  there  in  health.  The  normal  secretion  of  the  vagina 
has  a  bactericidal  influence  which  may  be  attributed  in  part 
to  its  acidity.  The  upper  part  of  the  normal  cervix  uteri  is 
sterile,  while  bacteria  are  present  in  the  lower  part. 

According  to  Doderlein  the  properties  of  the  vaginal  secretion  are 
due  to  bacilli  which  very  commonly  occur  in  it.  The  secretion  is  most 
abundant  and  important  during  pregnancy.1 

TJ.  W.  Williams,  "Obstetrics,  A  Text-Book,  etc.,"  1903,  pp.  34,  773- 
7/5.  Wadsworth,  American  Journal  of  Obstetrics,  Vol.  XLIIL,  1901. 


154  MANUAL    OF    BACTERIOLOGY. 

The  smegma  of  the  external  genitals  contains  numerous 
bacteria,  among  which  are  frequently  found  bacilli  which 
retain  their  color  after  treatment  with  acids  in  the  Gabbett 
method  for  staining  tubercle  bacilli.  It  is  uncertain  whether 
these,  bacilli  form  a  special  group  of  organisms  by  them- 
selves, having  as  one  of  their  properties  the  power  of  re- 
taining the  stain  after  acids,  or  whether  they  are  bacilli  of 
no  particular  sort,  which  resist  acids  after  staining  owing 
to  the  oily  material  with  which  they  have  been  impregnated 
in  this  peculiar  secretion.  These  organisms  must  be  taken 
into  account  in  staining  for  tubercle  bacilli,  urine  or  other 
secretions  which  might  accidentally  contain  particles  of 
smegma.  Usually  the  employment  of  alcohol  after  the  acid 
will  remove  the  color  from  the  smegma  bacilli  (Hueppe). 
Sometimes  smegma  bacilli  are  as  resistant  as  tubercle  bacilli 
to  decolorizing  agents  (Welch)  ;  see  page  44.  Similar  acid- 
proof  bacilli  occur  about  the  genitals  of  the  domestic  ani- 
mals.1 

The  bacteria  of  the  stomach  and  intestines  are  of  great 
interest  and  importance.  The  alimentary  tract  of  new-born 
infants  and  the  meconium  are  sterile.  In  from  four  to 
eighteen  hours  organisms  begin  to  appear.  They  may 
enter  either  from  the  mouth  or  the  anus.  There  seems  to 
be  no  constancy  in  the  nature  of  the  forms  which  are  found 
at  first,  but  their  character  depends  upon  the  surroundings. 

The  bacterial  inhabitants  of  the  stomach  are  less  constant 
than  we  shall  find  those  of  the  intestines  to  be.  Under 
normal  circumstances  they  seem  to  be  those  introduced 
from  the  mouth.  Different  investigators,  at  all  events, 
have  met  with  quite  different  species.  It  appears  that  the 
hydrochloric  acid  (about  2  parts  per  thousand)  present  in 
the  gastric  juice  at  the  height  of  digestion  possesses  decided 
germicidal  properties.  This  germicidal  power  exercises  a 
restraining  influence  upon  fermentation  due  to  bacteria,  and 
1  Cowie,  Journal  Experimental  Medicine,  Vol.  V.,  p.  205. 


THE    BACTERIA    OF    THE   NORMAL    HUMAN    BODY.      155 

probably  serves  as  a  safeguard  against  the  introduction  of 
pathogenic  germs  into  the  intestines.  That  is  particularly 
important  in  the  case  of  the  spirillum  of  cholera,  which  is 
excessively  sensitive  to  the  action  of  acids.  Nevertheless 
many  bacteria  are  able  to  reacrTthe  intestines  uninjured,  as 
the  acidity  of  the  gastric  juice  does  not  reach  its  height 
until  some  hours  after  eating.  Such  bacteria  will  be  those 
which  are  most  resistant  and  those  which  form  spores.  In 
the  intervals  when  hydrochloric  acid  is  absent  from  the 
stomach,  lactic  acid  appears.  It  is  formed  from  carbohy- 
drates by  a  large  number  of  species  of  bacteria.  In  con- 
ditions of  fermentation,  sarcina  ventriculi  and  yeasts  may 
be  present  in  large  numbers;  in  the  healthy  stomach  they 
occur  in  much  smaller  numbers. 

The  intestine  of  the  infant  in  whom  feeding  has  become 
well  established  was  found  by  Escherich  to  contain  two 
principal  species  of  bacteria — in  the  lower  part  of  the  in- 
testine the  bacillus  coli  communis,  in  the  upper  part  the 
bacillus  lactis  aerogenes.  More  recently  it  has  been  shown 
that  the  stools  of  milk-fed  infants,  and  to  a  less  extent  of 
adults,  contain  large  numbers  of  anaerobic  bacilli,  which 
stain  by  Gram's  method  (bacillus  bifidus — Tissier,  bacillus 
acidophilus — Moro).  These  bacteria  have  not  been  fully 
studied.1 

The  number  of  bacteria  in  a  milligram  of  human  fecal 
matter  has  been  estimated  at  from  seventy  thousand  to 
thirty-three  million.  It  is  estimated  that  about  one-third  of 
the  fecal  matter  of  adults  consists  of  bacteria.  The  small 
intestine  of  adults  has  been  found  by  different  observers  to 
contain  very  different  species.  The  majority  of  these  ap- 
pear to  have  been  introduced  from  the  mouth  in  food  or 
water.  The  bacillus  coli  communis,  however,  occurs  in- 
variably in  health  not  only  in  the  intestine  of  man,  but  also 

1  Metchnikoff,  "  Les  Microbes  Intestinaux,"  Bulletin  dc  I'Institut  Pas- 
teur, May  15  and  30,  1903. 

14 


156  MANUAL    OF    BACTERIOLOGY. 

in  that  of  many  animals,  especially  in  the  lower  part.1     The 
pyogenic  micrococci  very  often  occur  in  the  intestine. 

In  the  case  of  ruminant  animals  like  the  cow  and  sheep, 
the  decomposition  of  cellulose,  which  forms  so  large  a  part 
of  their  food,  appears  to  be  effected  by  bacteria.  Bacteria 
having  this  power  are  constantly  found  in  the  stomachs  of 
ruminants.  The  best  known  species  is  that  called  bacillus 
amylobacter.  It  is  questionable  whether  the  products  of 
the  decomposition  of  cellulose  have  any  nutritive  value. 

Pasteur  some  years  ago  expressed  the  opinion  that  if  animals  could 
be  placed  in  such  surroundings  that  bacteria  could  be  excluded  from  the 
alimentary  canal  and  the  food,  life  would  be  impossible.  This  view 
has  excited  much  controversy,  and  was  apparently  disproved  by  the 
experiments  of  Nuttall  and  Thierfelder.  These  investigators  succeeded 
in  removing  guinea-pigs  from  the  mother  by  Caesarean  section,  and  in 
keeping  them  alive  in  sterile  surroundings,  upon  sterile  food,  so  that  the 
contents  of  the  alimentary  canal  remained  sterile.  Schottelius,  who 
worked  with  chickens,  obtained  contrary  results,  however,  so  that  this 
interesting  question  is  still  undecided. 

1  Moore  and  Wright,  "  Bacillus  coli  communis  from  Certain  Species 
of  Domesticated  Animals,"  American  Medicine,  March,  1902. 


BACTERIA    IN    DISEASE.  157 


CHAPTER  V. 

BACTERIA    IN    DISEASE. 

To  the  physician  and  the  student  of  medicine  the  study  of 
bacteriology  is  interesting  chiefly  on  account  of  the  great 
importance  attributed  to  bacteria  in  producing  disease. 
The  presence  in  an  organism  of  one  or  a  number  of  organ- 
isms of  another  species,  which  flourish  as  parasites  upon 
the  first,  is  a  phenomenon  of  very  wide  occurrence  in  na- 
ture. It  is,  in  fact,  nearly  universal.  It  may  be  observed 
among  plants  as  well  as  animals,  for  example  in  the  familiar 
galls  seen  on  some  of  the  higher  plants,  and  mostly  caused 
by  the  larvae  of  insects  harbored  by  the  plant.  We  also 
find  animals,  such  as  tape-worms  and  the  trichina  spiralis, 
living  as  parasites  upon  other  animals.  The  functions  of 
the  bacteria  make  them  peculiarly  suited  to  leading  a  para- 
sitic existence.  The  fact  that  they  possess  no  chlorophyll, 
and  that  they  are  therefore  unable  to  form  carbon  com- 
pounds from  the  carbon  dioxide  of  the  atmosphere,  makes 
it  necessary  for  them  to  secure  such  compounds  from  pre- 
existing organic  matter.  Many  of  them,  furthermore, 
flourish  better  when  they  are  able  to  obtain  nitrogenous 
food  from  organic  matter  rather  than  from  inorganic  salts 
containing  nitrogen.  Most  bacteria  find  the  necessary  nutri- 
ment in  the  dead  bodies  of  other  animals  and  plants ;  they 
constitute  what  are  known  as  saprophytes.  But  some  of 
them  flourish  upon  the  living  bodies  of  other  plants  and 
animals  in  whom  they  may  produce  disease. 

The  phenomena  of  disease,  we  shall  find,  are  very  largely 
due  to  the  numerous  waste  products  of  the  activities  of 
bacteria,  which  act  as  poisons  to  the  host. 


158  MANUAL    OF    BACTERIOLOGY. 

The  diseases  of  plants  known  to  be  caused  by  bacteria 
are  not  very  numerous.  Among  them  may  be  mentioned 
pear-blight,  due  to  micrococcus  amylovorus.1  Among  lower 
animals  bacteria  very  frequently  produce  diseases — for 
example,  chicken-cholera,  symptomatic  anthrax,  erysipelas 
of  swine,  hog-cholera,  tuberculosis,  anthrax  and  glanders. 

Koch  formulated  certain  rules  which  he  considered  must 
be  complied  with  in  order  to  p.ove  that  any  microorganism 
was  the  cause  of  a  particular  disease : 

First.  That  the  organism  should  always  be  found  micro- 
scopically in  the  bodies  of  animals  having  the  disease;  that 
it  should  be  found  in  that  disease  and  no  other;  that  it 
should  occur  in  such  numbers  and  be  distributed  in  such  a 
manner  as  to  explain  the  lesions  of  the  disease. 

Second.  That  the  organism  should  be  obtained  from  the 
diseased  animal  and  propagated  in  pure  culture  outside  of 
the  body. 

Third.  That  the  inoculation  of  these  germs  in  pure  cul- 
tures, which  had  been  freed  by  successive  transplantations 
from  the  smallest  particle  of  matter  taken  from  the  original 
animal,  should  produce  the  same  disease  in  a  susceptible 
animal. 

Fourth.  That  the  organism  should  be  found  in  the  lesions 
thus  produced  in  the  animal. 

An  infectious  disease  is  a  disease  which  is  caused  by  a 
microorganism  growing  in  the  body  of  the  animal  having 
the  disease.  Such  microorganisms  are  usually  bacteria, 
but  not  always;  for  example,  malaria  is  produced  by  a 
minute  animal  organism. 

A  contagious  disease  is  one  which  is  acquired  from  an 
individual  having  the  disease.  Most  contagious  diseases 
are  infectious,  but  infectious  diseases  are  not  necessarily 
contagious.  The  words  are  often  used  very  loosely,  and 

1  See  E.  Smith  in  Ccntralblatt  f.  Baktcriologic,  etc.,  Z  \\eite  Abtheilung, 
TU1.  V.,  p.  271 ;  Bd.  VII.,  p.  88. 


BACTERIA    IN    DISEASE.  159 

it  is  no  longer  possible  or  very  desirable  to  draw  the  line 
sharply  between  them.  Fomites  are  the  materials  on  which 
the  contagious  element  is  conveyed. 

A  miasmatic  disease  is  a  variety  of  infection  in  which 
the  microorganisms  are  not  received  from  another  case  of 
the  disease,  but  are  derived  from  the  external  world,  par- 
ticularly through  foul  air. 

The  following  is  a  list  of  the  most  important  diseases  of 
man  caused  by  bacteria.  The  proof  as  required  by  the 
rules  of  Koch  is  not  complete  for  all  of  them : 

Tuberculosis,  Gonorrhea, 

Leprosy,  Chancroid  or  soft  chancre, 

Glanders,  Lobar  pneumonia, 

Anthrax,  Influenza, 

Tetanus,  Diphtheria, 

Malignant  edema,  Typhoid  fever, 

Bubonic  plague,  Dysentery  (not  amcebic  dys- 

Malta  Fever,  entery), 

Suppuration      and  certain     Asiatic  cholera, 
inflammatory      conditions      Relapsing  fever, 

allied  to  it,  Rhinoscleroma  (?), 

Erysipelas,  Actinomycosis. 

Malaria  and  amoebic  dysentery  are  caused  by  microscopic 
animal  organisms  (protozoa).  It  has  been  claimed  that 
small-pox  is  caused  by  protozoa;  this  view  has  acquired 
added  interest  from  the  recent  researches  of  Councilman. 
Recent  work  indicates  that  the  "  sleeping  sickness  "  (of 
Africa)  and  yellow  fever  may  be  caused  by  protozoa  (see 
appendix).  Thrush  and  certain  parasitic  skin  diseases  are 
caused  by  fungi  of  more  highly  organized  structure  than 
bacteria. 

In  each  of  the  following  diseases  there  is  good  reason  to 
think  that  the  cause  is  some  kind  of  microorganism,  but  it 
has  not  yet  been  discovered  : 


l6o  MANUAL    OF    BACTERIOLOGY. 

Syphilis,  Mumps, 

Chicken-pox,  Whooping-cough, 

Measles,  Yellow  fever, 

Scarlet  fever,  Typhus  fever, 

German  measles,  Hydrophobia, 

Dengue. 

Rheumatic  fever  and  Beri-beri  would  be  placed  in  this 
list  by  many  writers. 

The  causes  of  these  diseases  have  been  very  carefully 
sought  for  by  ordinary  bacteriological  methods  with  inde- 
cisive results.  Some  of  them  no  doubt  are  due  to  bacteria. 
In  recent  years  numerous  observers  have  described  a  diplo- 
coccus  or  short  streptococcus  as  the  cause  of  rheumatic  fever 
or  acute  rheumatism.  In  the  case  of  yellow  fever  Sanarelli 
described  an  organism  (bacillus  icteroides)  as  its  cause,  but 
his  view  is  not  upheld  by  most  of  those  who  have  worked 
on  yellow  fever.1  The  bacillus  described  by  a  number  of 
observers  as  having  been  found  in  cases  of  whooping-cough 
may  also  be  the  cause  of  that  disease.2  Bacilli  have  also 
been  described  in  cases  of  measles  on  several  occasions. 
Lustgarten  has  described  a  bacillus  found  in  the  lesions  of 
syphilis  which  resembles  tubercle  and  smegma  bacilli.  More 
recently  Joseph  and  Piorkowsky3  have  cultivated  another 
bacillus  from  cases  of  syphilis ;  how  much  importance  should 
be  attached  to  it  cannot  as  yet  be  stated.  It  is  likely  that 
for  some  of  the  diseases  mentioned  other  procedures  than 
the  usual  methods  of  research  will  have  to  be  devised  in 
order  that  the  cause  may  be  discovered.  The  protozoa  may 
play  a  part  in  the  etiology  of  some  of  them.  Roux  believes 

1  Sanarelli,  La  Semainc  Medicare,  April  4,   1900.     Reed  and  Carroll, 
Journal  Experimental  Medicine,  Vol.  V. 

2  See  Czaplewski,   Ccniralblatt  f.   Baktcnologic,  Bd.  XXIV.,   1898,   p. 
865. 

3Ccutralb!att  f.  Bakteriologie,  Vol.  XXXI.,  1902,  Orig.  p.  445.  Ber- 
liner klin.  Wochcnschrift,  1902,  Nos.  13  and  14. 


BACTERIA    IN    DISEASE.  l6l 

that  contagious  pleuro-pneumonia  of  cattle  is  due  to  a 
microbe  so  minute  that  it  is  barely  visible  with  the  highest 
powers  of  the  microscope,  so  that  its  outlines  and  its  mor- 
phology can  not  be  studied.  The  virus  of  this  disease  re- 
mains virulent  after  being  passed  through  a  Pasteur  filter, 
showing  that  it  is  small  enough  to  go  through  its  pores. 
Similar  experiments  have  succeeded  with  a  number  of  other 
affections  of  animals  (of  which  the  best  known  is  foot  and 
mouth  disease) .  The  virus  may  pass  through  a  Pasteur  or 
Berkenfeld  filter  of  a  certain  coarseness,  but  is  restrained 
by  one  sufficiently  fine.  The  most  important  of  the  diseases 
in  this  class  is  yellow  fever.  Reed  and  Carroll  found  that 
the  infective  agent  of  yellow  fever  is  in  the  blood,  and  that 
the  serum  could  produce  yellow  fever  in  a  non-immune 
person  after  filtration  through  a  Berkenfeld  filter.1  These 
facts  suggest  the  possibility  that  failure  to  find  the  causes 
of  some  other  diseases  may  lie  in  the  fact  that  their  organ- 
isms are  so  small  as  to  be  nearly  or  entirely  invisible  to  the 
microscope. 

Modes  of  Introduction. — There  are  various  avenues  by 
which  bacteria  may  enter  the  body  to  produce  disease. 
Infection  of  the  embryo  through  the  ovum  or  semen  seems 
to  be  of  rare  occurrence.  Syphilis  (which  may  not  be  due 
to  bacteria)  is  transmitted  in  this  manner.  The  embryo 
may  be  infected  through  the  placenta,  although  not  com- 
monly. The  bacilli  of  typhoid  fever  and  the  pus-forming 
bacteria  have  been  known  to  be  conveyed  through  it. 
Tuberculosis  may  also  be  transmitted  through  the  placenta, 
how  frequently  is  still-  uncertain.  The  comparatively  com- 
mon occurrence  of  endocarditis  on  the  right  side  of  the 
heart  in  the  fetus  may  be  due  to  placental  infection.  Oc- 

1  See  Reed  and  Carroll,  American  Medicine,  February  22,  1902.  For 
an  admirable  review  of  this  subject  see  Roux,  "  Sur  les  Microbes  dits 
'  Invisibles,' "  Bulletin  de  I'Institut  Pasteur,  Vol.  I.,  Nos.  I  and  2. 


1 62  MANUAL    OF    BACTERIOLOGY. 

casionally  the  exanthematous  fevers  are  transmitted  from 
the  mother  to  the  fetus. 

The  surfaces  covered  with  thick  stratified  epithelium  are 
not  likely  to  be  penetrated  by  bacteria  excepting  by  direct 
introduction  through  some  wound  or  other  lesion.  This, 
for  instance,  is  true  of  the  skin,  the  mouth,  the  vagina  and 
bladder.  The  infection  of  bubonic  plague  appears  to  be 
introduced  most  often  by  means  of  wounds  in  the  skin. 
Bacteria  more  easily  penetrate  surfaces  having  a  thin 
columnar  epithelium  such  as  occurs  in  the  intestines,  the 
middle  ear,  bronchi  and  bronchial  tubes,  uterus  and  Fallo- 
pian tubes. 

The  thin,  flat  epithelial  cells  of  the  air-vesicles  of  the 
lungs,  as  would  be  expected,  seem  to  be  passed  with  com- 
parative ease.  On  epithelial  surfaces  covered  with  cilia, 
as  in  the  bronchi  and  bronchial  tubes,  the  Eustachian  tubes, 
the  uterus  and  Fallopian  tubes,  the  current  toward  the  ex- 
terior created  by  the  cilia  acts  beneficially  in  removing 
bacteria. 

The  tonsils  and  lymph-follicles  of  the  intestines,  espe- 
cially the  lymphoid  tissue  of  the  ileum  and  the  vermiform 
appendix,  are  points  where  bacterial  invasion  frequently 
begins.  The  lymphoid  tissue  of  the  appendix  may  have 
some  influence  in  predisposing  to  infection  at  that  point 
and  to  appendicitis.  On  the  other  hand,  it  is  certain  that 
the  progress  of  many  infections  is  checked  by  the  lymph- 
nodes.  That  is  repeatedly  seen  in  the  ordinary  post-mor- 
tem wound  where  the  spread  of  the  inflammation  along  the 
arm  is  checked  suddenly  at  the  elbow  or  axilla.  The  par- 
ticipation of  the  lymphoid  structures  in  most  infections  is 
well  known.  How  far  this  is  a  conservative  process  it  is 
impossible  to  say. 

In  most  cases  of  infectious  disease  a  point  of  entrance 
for  the  bacteria  may  be  discovered.  As  a  rule,  the  invading 


BACTERIA    IN    DISEASE.  163 

microbes  produce  a  lesion  at  the  point  where  they  are  in- 
troduced, as  in  the  familiar  cases  of  boils  and  carbuncles 
when  pyogenic  bacteria  enter  the  skin,  or  of  the  tubercles 
found  in  the  lungs  when  the  bacilli  lodge  in  the  respiratory 
tract.  However,  there  are  cases  of  septicemia  and  pyemia 
in  which  the  most  careful  search  fails  to  reveal  the  place  at 
which  the  bacteria  entered.  The  bacilli  of  plague  usually 
produce  no  reaction  at  the  point  of  entrance. 

It  is  probable  that  tubercle  bacilli  may  pass  through  thin 
epithelial  surfaces  and  lodge  in  the  deeper  structures  un- 
derneath, where  they  produce  definite  lesions.  For  exam- 
ple, they  may  pass  by  the  lungs  and  enter  the  bronchial 
glands,  and  form  tubercles  in  that  situation. 

Experiments  on  animals  have  shown  that  bacteria  may 
be  very  rapidly  disseminated  after  their  introduction.  The 
inoculation  of  mice,  for  instance,  with  anthrax  bacilli  has 
been  known  to  prove  fatal,  although  the  wound  was  washed 
immediately  with  the  strongest  antiseptic  solutions  or  the 
part  amputated  within  a  few  minutes. 

The  manner  in  which  infectious  agents  reach  human 
beings  varies  considerably.  Generally  speaking,  the  most 
important  element  will  be  found  to  be  direct  or  indirect 
connection  with  another  case  of  the  same  disease.  W.  H. 
Park  was  able  to  cultivate  diphtheria  bacilli  from  bed  cloth- 
ing soiled  by  the  expectoration  of  diphtheria  cases.  Bald- 
win has  shown  that  tubercle  bacilli  may  be  found  on  the 
hands  of  patients  having  pulmonary  tuberculosis,  especially 
those  who  expectorate  on  handkerchiefs. 

Excepting  under  certain  special  conditions,  the  Air  will 
not  contain  the  germs  of  disease.  The  dried  pulverized 
sputum  of  cases  of  pulmonary  tuberculosis  may  float  in  the 
atmosphere  as  dust  which  will  contain  tubercle  bacilli. 
Fliigge  states  that  powerful  expiratory  efforts  like  coughing 
and  sneezing  may  carry  tubercle  bacilli  with  small  particles 


164  MANUAL    OF    BACTERIOLOGY. 

of  secretion  into  the  air,  and  that  they  may  remain  in  sus- 
pension some  time.  The  pus-producing  bacteria  may  be 
present  in  dust.  Infectious  elements  which  happen  to  be 
present  in  the  air  will  usually  be  attached  to  particles  of 
dust.  Wool-sorter's  disease  is  a  name  sometimes  applied 
to  anthrax  in  man  when  acquired  by  those  engaged  in  the 
work  of  handling  wool,  and  in  which  the  anthrax  bacilli 
or  spores  may  be  conveyed  to  the  lungs  in  dust. 

The  atmosphere  in  the  vicinity  of  cases  of  the  exanthem- 
atous  fevers  at  times  probably  contains  the  germs  of  these 
diseases. 

Water  is  the  usual  medium  for  the  transmission  of  the  in- 
fection in  typhoid  fever,  and  Asiatic  cholera,  and  probably 
all  forms  of  dysentery. 

Milk  from  tuberculous  cows  may  carry  the  bacilli  of 
tuberculosis;  it  is  of  most  importance  in  the  case  of  young 
infants.  Typhoid  fever  and  cholera,  and  probably  scarlet 
fever  and  diphtheria  are  sometimes  conveyed  through  the 
medium  of  milk.  Bacteria  may  reach  the  intestines  in 
uncooked  food,  fruit  and  vegetables. 

The  Soil  is  of  importance  in  connection  with  tetanus  and 
malignant  edema,  whose  bacteria  are  frequently  found  in 
soil.  Bacillus  aerogenes  capsulatus  may  occur  in  the  soil, 
and  may  infect  dirty  wounds.  The  spores  of  anthrax  bacilli 
are  present  in  the  soil  of  certain  localities,  and  may  produce 
anthrax  in  cattle. 

Flies  and  other  insects  and  related  animals  are  capable 
of  carrying  the  bacteria  of  disease.  Under  suitable  condi- 
tions flies  play  an  important  part  in  transporting  the  bac- 
teria of  cholera  and  typhoid  fever  from  the  excreta  of  these 
diseases  to  food  substances,  which  they  may  contaminate. 
To  what  extent  diseases  are  disseminated  by  fleas,  bed- 
bugs and  similar  creatures  is  still  uncertain.1 

'Nnttall,  "Role  of  Insects,  etc.,  in  Disease,"  Johns  Hopkins  Hospit.-il 
Reports,  Vol.  VIII.,  1900. 


BACTERIA    IN    DISEASE.  165 

In  this  connection  it  is  proper  to  refer  to  certain  diseases 
due  to  animal  microorganisms.  Malaria  is  conveyed  from 
man  to  man  by  mosquitoes  of  the  genus  Anopheles,  and 
is  probably  transmitted  exclusively  in  this  manner.  The 
parasite  of  malaria  undergoes  part  of  its  cycle  of  develop- 
ment in  man,  and  another  part  in  the  mosquito.  Similarly, 
in  Texas  Fever,  a  disease  of  cattle,  it  has  been  shown  by 
T.  Smith  that  the  parasite  (a  protozoon,  Piroplasma)  passes 
from  cow  to  cow  through  the  cattle-tick  (Boophilus  annul- 
atus  or  bovis).1  In  surra,  a  disease  chiefly  affecting  horses, 
and  in  the  tsetse-fly  disease  of  animals  the  parasite  (a  proto- 
zoon, Trypanosoma ) ,  is  transmitted  by  the  bites  of  flies.2 
It  has  recently  been  shown  that  the  infectious  agent  of 
yellow  fever  may  be  introduced  into  man  by  mosquitoes  of 
the  genus  Stegomyia.  Under  the  administration  of  the 
United  States  Army  yellow  fever  was  suppressed  in  Havana 
chiefly  by  measures  intended  to  prevent  the  disease  from 
being  carried  by  mosquitoes.3 

Auto-Infection. — It  is  possible  for  the  bacteria  of  disease 
to  be  derived  from  the  individual's  own  body — auto-infec- 
tion. The  microbes  of  lobar  pneumonia,  for  instance, 
flourish  in  the  mouths  of  a  large  number  of  people.  The 
bacillus  coli  communis,  which  constantly  inhabits  the  in- 
testines, may  invade  other  organs  and  exhibit  pathogenic 
properties  when  the  way  is  opened  up  for  it  by  other  dis- 
ease processes. 

Bodily  Conditions  that  Dispose  to  Infection. — The  de- 
velopment of  an  infectious  disease  may  be  favored  by  cer- 
tain bodily  conditions.  Hunger,  cold  and  exhaustion  make 
the  body  more  liable  to  the  inroads  of  pathogenic  bacteria ; 
so  also  do  anemia  and  chronic  diseases.  Those  suffering 
from  diabetes,  as  is  well  known,  are  especially  liable  to 

1  See  V.  A.  Moore,  "  Infectious  Diseases  of  Animals,"  1902. 

2  Report  on  Surra,  U.  S.  Bureau  Animal  Industry,  1902. 

3  Carroll,    Journal   American    Medical   Association,    May   23,    1903. 


1 66  MANUAL    OF    BACTERIOLOGY. 

infection  by  the  pus-producing  bacteria  and  the  bacillus 
tuberculosis.  Dr.  Roswell  Park  believes  that  prolonged 
anesthesia  makes  patients  who  have  undergone  operations 
more  liable  to  surgical  infections,  and  that  absorption  of 
bacterial  poisons  and  auto-intoxication  due  to  the  products  of 
disordered  metabolism  of  the  patient's  own  cells,  predis- 
pose to  infection.  Some  of  the  above-mentioned  conditions 
can  be  imitated  in  laboratory  experiments.  Hens  in  a  nor- 
mal condition  are  not  susceptible  to  the  anthrax  bacillus,  but 
Pasteur  succeeded  in  making  them  contract  anthrax  by 
artificially  cooling  them.  Frogs,  on  the  other  hand,  which 
also  are  resistant  to  anthrax,  may  be  made  susceptible  by 
keeping  them  at  an  abnormally  high  temperature.  Rats 
were  made  more  susceptible  to  anthrax  by  physical  ex- 
haustion produced  by  making  them  run  a  treadmill,  and 
pigeons  by  starvation. 

Abbott  found  "  that  the  normal  vital  resistance  of  rabbits 
to  infection  by  streptococcus  pyogenes  is  markedly  dimin- 
ished through  the  influence  of  alcohol,  when  given  daily  to 
the  stage  of  acute  intoxication."  It  was  less  noticeable  for 
bacillus  coli  communis,  and  not  observed  for  staphylococcus 
pyogenes  aureus.  Pigeons  and  other  animals  have  been 
made  susceptible  to  anthrax  by  intoxicating  doses  of  alcohol. 

Climate  and  altitude  appear  to  influence  the  liability  to 
infection  with  the  tubercle  bacillus,  which  occurs  less  com- 
monly in  Colorado  and  some  other  elevated  regions  than 
in  lower  and  more  densely  populated  districts. 

Age. — In  general,  infants  are  more  susceptible  to  infec- 
tions than  adults,  though  apparently  nearly  exempt  from 
the  exanthematous  fevers  during  the  early  weeks  of  life. 
Osteomyelitis  is  commoner  in  infants  than  in  adults,  as  also 
is  tuberculous  meningitis. 

How  much  influence  is  to  be  ascribed  to  individual  pre- 
disposition in  contracting  or  warding  off  infection  is  unccr- 


BACTERIA    IN    DISEASE.  167 

tain.  Welch  says,  "  the  fact  that  some  individuals  are 
attacked,  and  others,  apparently  equally  exposed  to  the 
danger  of  infection,  escape,  is  not  always  due  to  any  espe- 
cial predisposition  on  the  part  of  the  former.  It  may  be 
that  the  germs  hit  the  one  and  miss  the  other,  and  we 
would  have  no  more  right  to  say  that  the  former  are  espe- 
cially predisposed  than  to  say  that  those  who  fall  in  battle 
are  predisposed  to  bullets  and  those  who  escape  are  bullet- 
proof." It  is  probable  that  the  importance  of  an  hereditary 
tendency  to  certain  infections,  notably  tuberculosis,  has  been 
overrated. 

Race. — The  influence  of  racial  predisposition  is  undeni- 
able. For  example,  it  is  known  that  the  negro  race  is  much 
less  susceptible  to  yellow  fever  than  the  white  race. 

Local  conditions  often  have  a  most  important  influence 
in  determining  the  occurrence  of  infections.  In  endocar- 
ditis the  lesion  usually  occurs  along  the  line  of  closure  of 
the  heart- valves,  indicating  that  the  point  subjected  to  the 
greatest  friction  is  the  part  of  the  endocardium  most  liable 
to  infection.  Regions  where  there  is  passive  hyperemia 
are  more  vulnerable,  as  is  seen  in  hypostatic  pneumonia. 
Localities  which  have  suffered  from  previous  inflammation 
or  irritation  are  rendered  more  liable  to  subsequent  infec- 
tion, as  when  the  bladder  or  pelvis  of  the  kidney  contain- 
ing a  calculus  becomes  the  seat  of  a  suppurative  cystitis  or 
pyelitis. 

Local  conditions  become  of  great  importance  in  surgery. 
The  surgeon  can  seldom  be  certain  of  dealing  with  a  per- 
fectly aseptic  wound,  and  must  rely  to  a  large  extent  upon 
the  power  inherent  in  the  fluids  and  tissues  to  prevent  the 
development  of  bacteria.  It  is  important,  therefore,  to  keep 
the  resisting  power  of  the  tissues  at  the  highest  possible 
point.  Injury  of  the  tissues  disposes  the  part  to  infection; 
so  do  strangulation  and  necrosis.  In  operating,  it  is  to  be 


1 68  MANUAL    OF    BACTERIOLOGY. 

remembered  that  hyperemic  and  edematous  parts  are  more 
likely  to  become  infected;  so  also  are  anemic  regions.  An 
infarct  of  the  lung  which  was  originally  sterile  may  be  in- 
fected with  bacteria  through  inhalation,  and  undergo  sup- 
puration or  gangrene.  The  presence  of  foreign  bodies  in 
the  tissues  disposes  to  infection.  Injection  of  the  staphy- 
lococcus  pyogenes  aureus  into  a  rabbit's  tissues  is  not  always 
followed  by  suppuration,  but  if  a  foreign  body,  like  a  piece 
of  sterilized  potato,  be  inserted  at  the  same  time,  infection 
is  much  more  likely  to  occur.  When  lesions  are  produced  in 
the  internal  viscera  of  animals  by  cauterization  or  crushing 
and  bacteria  then  injected  subcutaneously  or  into  the  blood, 
the  bacteria  lodge  in  the  lesions  and  multiply.1 

Amount  of  Infectious  Material. — A  large  number  of 
bacteria  introduced  into  the  body  simultaneously  will  be 
more  likely  to  produce  infection  than  a  small  number.  This 
factor  is  of  less  importance  with  organisms  whose  virulence 
is  very  constant  than  with  those  of  more  variable  virulence. 

Variability  in  the  Virulence  of  Bacteria. — The  occur- 
rence of  an  infectious  disease  depends  very  largely  upon 
the  virulence  of  the  bacteria.  Any  species  of  pathogenic 
bacteria  may  vary  in  virulence  at  different  times.  In  some 
cases  the  virulence  is  not  easily  lost,  as  with  the  anthrax 
bacillus;  in  others  the  virulence  is  maintained  in  cultures 
only  with  difficulty,  as  in  the  case  of  the  micrococcus  lan- 
ceolatus  (of  pneumonia)  and  the  streptococcus  pyogenes. 
As  a  rule,  the  virulence  is  likely  to  be  diminished  in  old 
cultures.  It  may  sometimes  be  preserved  better  in  the  ice- 
chest  than  at  the  room  temperature.  The  virulence  of  the 
anthrax  bacillus  becomes  diminished  if  it  is  cultivated  at 
42°  C.  Exposure  to  light  and  to  oxygen  tends  to  weaken 
the  virulence;  and  also  cultivation  upon  unfavorable  media, 
such  as  those  containing  a  small  proportion  of  carbolic  acid 
or  certain  other  chemical  germicides. 

1  Cheesman  and  Meltzer,  Journal  Experimental  Medicine,  Vol.  III. 


0 

BACTERIA    IN    DISEASE.  169 

In  laboratory  work  the  virulence  is  usually  maintained 
best  by  inoculating  the  bacteria  from  time  to  time  into  sus- 
ceptible animals.  Bacteria  coming  freshly  from  infected 
animals  are  likely  to  be  highly  virulent.  The  virulence 
may  be  increased  by  beginning  with  an  especially  sensitive 
animal  like  a  very  young  guinea-pig,  and  progressively  in- 
oculating into  less  sensitive  animals.  The  infection  of  rela- 
tively insusceptible  animals  may  sometimes  be  produced  by 
the  injection  of  a  very  large  dose  of  the  bacteria.  The 
addition  of  the  toxic  products  of  the  bacteria,  which  may 
be  obtained  by  using  large  doses  of  cultures  in  bouillon, 
makes  infection  more  likely.  Cultivation  on  a  particular 
medium  may  maintain  or  increase  the  virulence. 

Finally,  the  combination  of  two  or  more  kinds  of  bac- 
teria may  produce  infection  when  neither  one  would  do  so 
alone.  On  the  other  hand,  it  is  said  that  the  fatal  effects 
of  an  inoculation  of  virulent  anthrax  bacilli  into  a  sus- 
ceptible animal  may  be  averted  if  the  animal  be  inoculated 
with  a  culture  of  bacillus  pyocyaneus  shortly  afterward. 

Mixed  Infection. — It  is  not  uncommon  in  disease  to  find 
two  kinds  of  bacteria  associated  together,  producing  a 
mixed  infection.  In  diphtheria,  very  frequently,  the  ba- 
cillus of  diphtheria  is  found  to  be  accompanied  in  the  mem- 
brane by  the  streptococcus  pyogenes.  The  course  of  the 
diphtheria  may  be  modified  in  this  manner.  The  term 
secondary  infection  is  rather  loosely  used.  It  is  sometimes 
employed  to  designate  an  infection  occurring  in  an  indi- 
vidual, the  resisting  power  of  whose  tissues  has  been  weak- 
ened by  some  chronic  organic  disease.  Such  an  infection 
is  often  called  a  terminal  infection.  Terminal  infections 
are  very  common  in  cases  of  carcinoma,  chronic  nephritis, 
arteriosclerosis,  and  in  many  other  diseases. 

Concerning  terminal  infections  Osier  says :  "  It  may 
seem  paradoxical,  but  there  is  truth  in  the  statement  that 


MANUAL    OF    BACTERIOLOGY. 

persons  rarely  die  of  the  disease  with  which  they  suffer. 
Secondary  infections,  or,  as  we  are  apt  to  call  them  in 
hospital  work,  terminal  infections,  carry  off  many  of  the 
incurable  cases  in  the  wards." 

The  term  secondary  infection  is  also  used  for  the  modifi- 
cation of  an  infectious  process  which  has  been  in  existence 
for  some  time,  by  infection  with  a  second  variety  of  bacte- 
ria. That  takes  place,  for  instance,  in  pulmonary  tubercu- 
losis, when  the  invasion  of  the  already  tuberculous  lungs  by 
the  pyogenic  micrococci  assists  in  the  formation  of  cavities. 
In  this  sense  it  will  be  seen  that  the  term  secondary  infec- 
tion is  used  as  a  name  for  a  variety  of  mixed  infection.  In 
the  secondary,  mixed  and  terminal  infections,  the  bacteria 
which  enter  secondarily  are  likely  to  be  of  the  pus-producing 
varieties,  especially  the  streptococcus  pyogenes. 

As  to  the  mechanism  which  bacteria  make  use  of  in 
order  to  produce  disease,  according  to  our  present  knowl- 
edge, they  work  chiefly  through  the  poisonous  substances 
formed  by  them  and  deposited  in  the  bodies  of  the  persons 
suffering  from  the  disease.  The  theory  that  bacteria  have 
an  important  influence  through  the  destruction  of  substances 
taken  by  them  from  the  body  of  the  patient  for  food,  is  no 
longer  entitled  to  much  weight;  neither  are  we  able  in  most 
cases  to  account  for  the  phenomena  of  disease  by  any 
mechanical  action  on  the  part  of  the  bodies  of  bacteria. 
That  such  action  does  occasionally  take  place  may  be  seen 
in  experimental  anthrax  in  mice,  where  the  blood-capil- 
laries of  the  liver  and  kidneys  may  be  completely  plugged 
with  masses  of  anthrax  bacilli.  The  diseases  in  which 
the  circulating  blood  is  swarming  with  bacteria  are  much 
commoner  in  the  lower  animals  than  in  man. 

Toxemia. — By  toxemia  is  meant  the  absorption  of  poison- 
ous bacterial  products  from  a  localized  point  of  invasion, 
and  their  dissemination  throughout  the  body  by  means  of 


BACTERIA    IN    DISEASE. 

the  circulation.  We  see  typical  toxemias  in  diphtheria  and 
tetanus.  In  surgery  the  term  sapremia  is  used  to  cover  a 
similar  condition  of  affairs  when  the  absorption  proceeds 
from  a  wound  or  denuded  surface,  as  may  happen  in  the 
puerpural  uterus. 

Septicemia. — In  septicemia  there  is  not  only  absorption 
of  the  bacterial  poisons,  but  the  bacteria  have  invaded  the 
living  tissues.  Bacteriologists  usually  employ  the  word 
septicemia  to  describe  the  wide  dissemination  of  bacteria 
through  the  body  and  the  presence  of  a  large  number  of 
them  in  the  circulating  blood.  In  this  sense  septicemias 
are  commoner  in  lower  animals  than  in  man;  anthrax  and 
infection  with  micrococcus  lanceolatus  would  be  examples. 
Typical  septicemias  in  man  are  found  in  relapsing  fever  and 
certain  cases  of  bubonic  plague.  For  pyemia,  see  the  article 
on  Suppuration,  Part  IV. 

The  principal  agencies  in  effecting  recovery  from  in- 
fectious diseases  are  the  presence  or  formation  in  the  body 
of  substances  which  destroy  bacteria  (lysins),  the  develop- 
ment of  new  substances  which  also  neutralize  their  action 
(antitoxins),  and  their  destruction  by  the  cells  of  the  body 
(phagocytosis).  These  phenomena  are  discussed  in  the 
chapter  on  immunity.  A  factor  of  less  importance  is  the 
elimination  of  bacteria  by  the  excretory  organs.  Experi- 
ments on  animals  indicate  that  bacteria  which  have  been 
injected  into  the  body  do  not  appear  in  the  urine  until  they 
have  damaged  the  structure  of  the  kidney.  In  typhoid  fever, 
the  bacilli  of  typhoid  may  occur  in  the  urine  in  great  num- 
bers; the  condition  of  the  kidney  in  the  generality  of  such 
cases  has  not  thus  far  been  determined.  The  extent  to 
which  the  excretory  organs  act  in  eliminating  bacterial 
toxins  is  not  yet  known.  Some  bacteria,  as  has  already 
been  stated,  may,  in  the  end,  produce  substances  that  are 
inimical  to  their  own  growth. 
'5 


172  MANUAL    OF    BACTERIOLOGY. 


CHAPTER   VI. 

TOXINS.1 

IT  is  now  generally  believed  that  in  most,  if  not  all  of 
the  infectious  diseases,  the  principal  symptoms  and  lesions 
are  to  be  attributed  to  the  action  of  poisonous  substances 
formed  by  the  bacteria.  The  part  that  bacteria  play  can 
be  understood  best  by  recalling  the  work  of  the  saprophytes 
in  producing  fermentation  and  putrefaction.  It  has  already 
been  shown  that  the  poisoning  that  comes  from  eating  de- 
composed meat,  fish,  or  cheese  results  from  poisons  which 
bacteria  have  elaborated  in  the  course  of  their  growth. 
In  infectious  diseases  we  suppose  the  bacteria  to  grow  in- 
side of  the  body  and  to  form  their  poisons  in  it;  not  before 
their  introduction  into  it,  as  in  these  cases  of  poisoning  with 
spoiled  food.  If  it  were  possible  for  the  cells  of  ordinary 
yeast  to  grow  in  the  living  human  body  and  to  produce 
alcohol  from  the  grape-sugar  of  the  body-fluids,  the  person 
so  infected  might  be  expected  to  suffer  from  alcoholic  in- 
toxication as  long  as  the  infection  lasted.  This  impossible 
illustration  although  not  entirely  accurate  may  help  to  make 
clear  what  does  happen  in  an  infectious  disease  due  to  bac- 
teria, where  poisons  formed  in  a  manner  analogous  to  the  for- 
mation of  alcohol  produce  intoxications  analogous  to  alco- 
holic intoxication.  Certain  infectious  diseases  exhibit  the 
element  of  poisoning  by  bacterial  products  in  an  extremely 
marked  manner.  In  tetanus  the  local  wound  may  be  trifling, 
and  may  seem  utterly  incapable  of  having  given  rise  to  the 

1  For  a  full  consideration  of  this  subject  see  Vaughan  and  Novy, 
"  The  Cellular  Toxins,"  1902. 


TOXINS.  1/3 

violent  muscular  spasms  from  which  the  patient  suffers.  In 
diphtheria,  although  the  condition  in  the  throat  may  be  one 
of  severe  inflammation,  it  is,  of  itself,  insufficient  to  explain 
the  profound  prostration  and  other  symptoms  of  general 
poisoning  which  the  case  manifests. 

The  first  bacterial  poisons  to  be  studied  thoroughly  were 
those  called  ptomaines.  Observing  the  poisonous  effects 
which  follow  the  injection  into  animals  of  certain  ptomaines 
derived  from  bacterial  cultures,  it  was  suggested  that  similar 
ptomaines,  formed  by  the  action  of  bacteria  in  the  living 
body,  might  account  for  the  symptoms  of  many  of  the  in- 
fectious diseases.  The  ptomaines  were  most  readily  studied 
because  of  the  comparative  facility  with  which  they  could 
be  isolated  in  a  condition  of  purity,  where  their  exact 
chemical  nature  could  be  determined. 

"  A  Ptomaine  is  an  organic  chemical  compound,  basic  in 
character,  formed  by  the  action  of  bacteria  on  nitrogenous 
matter."  (VAUGHAN  and  Now.) 

The  peculiar  coloring  which  distinguishes  cultures  of  the 
bacillus  pyocyaneus  is  due  to  a  ptomaine  called  pyocyanin 
and  its  derivatives. 

A  group  of  substances  of  a  similar  nature  called  leuco- 
maines  has  been  discovered,  which  are  formed  within  the 
body  and  not  by  the  action  of  the  bacteria.  Leucomaincs 
may  then  be  defined  as  "  basic  substances  which  result  from 
tissue  metabolism  in  the  body."  (VAUGHAN  and  Now.) 

Further  study  has  demonstrated,  however,  that  the  char- 
acteristic features  of  the  infectious  diseases  are  not  due  to 
ptomaines.  Some  of  the  poisons  formed  by  bacteria  have 
been  described  as  albumens  and  have  given  rise  to  the 
name  toxalbumen.  It  appears,  however,  that  bacterial 
poisons  are  not  necessarily  of  an  albuminous  nature  either, 
and  at  the  present  time  it  seems  best  to  call  the  bacterial 
poisons  whose  chemical  nature  is  uncertain  simply  to.ruis. 


174  MANUAL    OF    BACTERIOLOGY. 

Substances  which  produce  effects  in  animals  similar  to  the 
bacterial  poisons  may  be  extracted  from  certain  plants, 
notably  abrin,  which  is  derived  from  the  jequirity  bean,  and 
ricin,  which  comes  from  the  castor-oil  bean.  The  venom 
of  poisonous  snakes,  which  is  elaborated  by  the  epithelial 
cells  of  certain  glands,  also  acts  in  much  the  same  manner. 

Bacterial  poisons  are  of  two  principal  sorts  :  ( i )  Some- 
times they  appear  to  be  formed  as  excretions  from  the  bac- 
teria. They  then  occur  in  solution  in  liquid  cultures,  and 
they  may  be  separated  from  the  bacteria  by  filtration. 
The  toxins  of  diphtheria  and  tetanus  are  typical  examples. 
(2)  The  poisons  of  most  bacteria  occur  chiefly  in  the  bodies 
of  the  bacterial  cells.  Such  toxins  are  called  intracellular. 
Those  of  typhoid  fever  and  cholera  are  examples.  Precisely 
how  intracellular  toxins  are  set  free  in  the  body  of  an  in- 
fected animal  is  not  clearly  understood.  It  is,  however, 
known  that  many  of  the  bacteria  in  an  infected  individual 
are  dead  and  disintegrated.  The  theories  concerning  toxins 
are  also  considered  in  the  next  chapter. 

Sometimes  the  results  of  the  injection  of  excessively 
small  doses  of  a  toxin  are  so  tremendous  as  to  have  given 
rise  to  the  suggestion  that  the  toxins  may  be  allied  to  the 
ferments,  like  pepsin  and  trypsin,  in  their  nature. 

Owing  to  the  instability  of  the  toxins  it  has  not  been 
possible  to  isolate  them  in  a  state  of  purity  so  as  to  deter- 
mine their  exact  chemical  character.  They  have,  never- 
theless, been  obtained  in  some  cases  in  an  extremely  con- 
centrated form.  Brieger  and  Conn  obtained  a  toxin  from 
tetanus  bacilli  of  which  .00000005  gram  killed  a  mouse 
weighing  15  grams.  Roux  and  Yersin  obtained  a  toxin 
from  diphtheria  bacilli  of  which  .00005  gram  was  capable  of 
killing  a  guinea-pig.  These  figures  indicate  a  capability 
for  poisoning  that  is  simply  inconceivable.  Such  proper- 
ties permit  bacteria  growing  in  a  comparatively  limited  area 
to  manifest  their  evil  effects  at  remote  parts  of  the  body. 


TOXINS.  175 

A  curious  and  unexplained  effect  of  some  toxins  is  the 
production  of  minute  areas  of  necrosis  in  certain  viscera,  as 
the  liver.  Such  "  focal  necroses  "  have  been  observed  to 
be  formed  by  the  poisons  of  the  bacilli  of  diphtheria,  of 
typhoid  fever,  and  of  the  micrococcus  lanceolatus  (of  pneu- 
monia), and  following  the  injection  of  abrin  and  ricin. 

Besides  the  poisonous  substances  produced  by  the  bacilli 
of  diphtheria  and  of  tetanus,  toxic  substances  have  been 
obtained  from  the  spirillum  of  cholera,  the  bacillus  of  ty- 
phoid fever,  the  bacillus  coli  communis,  the  bacillus  of 
bubonic  plague,  and  from  the  bacilli  of  tuberculosis  and 
glanders.  The  extract  from  cultures  of  tubercle  bacilli,  called 
tuberculin,  and  that  from  glanders  bacilli,  called  mallein, 
contain  toxins  produced  by  these  germs,  and  will  be  spoken 
of  in  connection  with  the  bacteria  themselves.  Vaughan1 
has  succeeded  in  cultivating  anthrax  bacilli,  colon  bacilli, 
and  other  bacteria  on  large  surfaces  of  solid  media,  so  as  to 
secure  quantities  of  the  bacterial  cells  sufficient  for  extensive 
chemical  tests.  The  toxin  of  the  colon  bacillus  proved  to  be 
a  very  stable  substance,  and  resistant  to  heat.  Most  toxins 
become  inactive  at  comparatively  low  temperatures.  (60° 
to  70°  C.) 

Prudden  and  Hodenpyl  found  that  the  injection  of  dead 
tubercle  bacilli  was  followed  by  the  development  of  lesions 
resembling  tubercles,  which,  of  course,  did  not  increase  in 
number  or  become  disseminated. 

There  is  good  reason  on  both  clinical  and  experimental 
grounds  to  believe  that  toxic  substances  are  formed  by  the 
micrococcus  lanceolatus  (of  pneumonia).  The  symptoms 
of  a  disease  as  it  occurs  in  man  cannot  be  imitated  in  any 
other  case  as  accurately  as  happens  after  the  injection  of  the 
toxins  of  tetanus  and  diphtheria  in  the  lower  animals. 

1  American  Medicine,  May  18,  1901.  Journal  American  Medical  Asso- 
ciation, March  28,  190.3. 


176  MANUAL    OF    BACTERIOLOGY. 


CHAPTER   VII. 

IMMUNITY. 

UNDER  the  title  of  immunity  a  number  of  nearly  related 
subjects  may  be  discussed.  It  has  already  been  shown  that 
the  body  is  constantly  liable  to  the  attacks  of  pathogenic 
microbes,  which  are  endeavoring  to  effect  an  entrance.  Some 
of  the  defences  of  the  body  have  been  described.  But  it  has 
been  found  that  there  are  other  more  subtle  and  at  the  same 
time  far  more  powerful  weapons,  which  usually  succeed  in 
repelling  the  invasion.  This  will  answer  the  question  often 
asked,  Why  do  we  not  all  constantly  have  infectious  dis- 
eases ?  In  case  bacteria  do  gain  a  foothold  in  the  body  and 
produce  what  we  call  an  infectious  disease,  we  have  to  con- 
sider the  means  by  which  the  intruders  are  overcome.  The 
exemption  from  a  second  visitation  which  often  follows  one 
attack  of  an  infectious  disease  also  needs  to  be  accounted 
for. 

Certain  facts  concerning  immunity,  some  of  which  were 
observed  many  years  ago,  are  extremely  interesting,  but 
very  difficult  of  explanation.  Even  in  the  light  of  recent 
bacteriological  researches  their  interpretation  is  by  no  means 
clear.  The  immunity  which  an  individual  who  has  suffered 
from  an  attack  of  measles  or  scarlet  fever  possesses  from  a 
second  attack  of  the  same  disease  is  well  known ;  so  also  is 
the  immunity  from  small-pox  which  is  conferred  by  vacci- 
nation. Such  an  immunity  is  called  "  acquired."  There  is 
also  a  "  natural  "  immunity.  Field-mice  are  susceptible  to 
glanders  and  house-mice  are  not.  House-mice  are  suscep- 
tible to  mouse-septicemia  and  field-mice  are  not.  Although 


IMMUNITY.  177 

sheep,  as  a  rule,  are  easily  infected  with  anthrax,  this  disease 
seldom  occurs  in  sheep  of  the  Algerian  variety  or  race.  The 
immunity  which  belongs  to  a  race,  but  not  to  a  whole  species, 
is  sometimes  called  "  racial." 

The  occurrence  of  immunity  from  a  second  attack  of  an 
infectious  disease  has  given  rise  to  numerous  hypotheses  in 
the  past.  One  theory  supposed  that  after  an  attack  of  the 
disease  certain  bacterial  products  are  retained  within  the 
body  which  prevent  a  second  invasion.  Another  theory 
supposed  that  the  attack  of  the  disease  exhausts  the  supply 
of  some  substance  necessary  for  the  growth  of  the  microbes, 
as  plants  sometimes  exhaust  the  soil  they  grow  in. 

It  will  be  best  to  deal  first  with  the  results  of  some  of  the 
experimental  attempts  to  produce  immunity.  The  more 
recent  theories  will  then  be  considered. 

Small-pox  and  Vaccination. — The  origin  of  vaccination 
against  small-pox  with  the  virus  of  cow-pox  has  been  de- 
scribed in  the  historical  sketch  (p.  21).  The  nature  of  the 
protection  furnished  by  this  virus  has  been  the  subject  of 
much  controversy.  The  opinion  of  the  present  day  inclines 
to  regarding  vaccinia  as  small-pox  which  has  been  modified 
by  passage  through  a  relatively  insusceptible  animal.  Cer- 
tainly there  are  many  analogies  between  the  protection 
against  small-pox  afforded  by  vaccination  and  the  other 
examples  of  artificial  immunity  mentioned  below. 

This  question  cannot  be  settled  with  certainty  until  the 
organisms  causing  small-pox  and  vaccinia  have  been  isolated 
in  pure  culture.  Their  identity  and  mode  of  action  may  then 
be  determined. 

Small-pox  has  been  inoculated  into  calves  and  passed 
through  other  calves  in  succession,  producing  finally  an 
eruption  indistinguishable  from  cow-pox.  Lymph  taken 
from  such  calves  has  been  used  successfully  to  vaccinate 
children.  Not  only  does  cow-pox  protect  against  small-pox, 


178  MANUAL    OF    BACTERIOLOGY. 

but  it  has  been  shown  that  small-pox  protects  against  cow- 
pox. 

Immunity  Produced  by  Inoculation  with  Bacteria  of 
Diminished  Virulence. — Pasteur  conceived  the  idea  of 
attenuating  the  virulence  of  the  bacilli  of  fowl-cholera  by 
prolonged  exposure  to  the  air.  He  made  use  of  the  atten- 
uated virus  as  a  vaccine  against  the  disease. 

A  nearly  similar  principle  was  shortly  afterward  applied 
by  him  to  the  preparation  of  a  vaccine  against  anthrax. 
When  anthrax  bacilli  were  cultivated  at  a  temperature  of 
43 °C.,  Pasteur  obtained  bacilli  of  very  slight  virulence. 
Such  bacilli  did  not  produce  death  when  inoculated  into 
animals  that  were  ordinarily  susceptible.  Yet  animals  that 
were  vaccinated  with  this  virus  were  able  afterward  to 
resist  inoculation  with  fully  virulent  anthrax  bacilli.  (See 
Bacillus  anthracis,  Part  IV.) 

In  the  case  of  erysipelas  of  swine  (French  rouget;  Ger- 
man, Schweinerothlauf)  Pasteur  secured  bacilli  of  dimin- 
ished virulence  by  injecting  virulent  bacilli  into  relatively 
insusceptible  animals.  The  animal  used  was  the  rabbit. 
The  bacilli  were  passed  through  several  rabbits  in  succes- 
sion. Cultures  taken  from  the  last  of  the  series  produced 
a  milder  form  of  the  disease  and  an  amount  of  immunity, 
the  value  of  which  is  in  dispute. 

In  still  another  disease,  black  leg  of  cattle  or  symptomatic 
anthrax  ( French,  charbon  sytnptomatique ;  German,  Rausch- 
brand),  an  attenuated  virus  is  secured  by  the  use  of  heat. 
The  pulp  from  the  infected  muscle  of  a  diseased  animal, 
containing  the  bacilli,  is  squeezed  from  it  and  heated  to  a 
temperature  of  95°  to  99°  C.  for  six  hours.  The  dried 
material  mixed  with  water  constitutes  the  vaccine.  The 
Department  of  Agriculture  of  the  United  States  now  fur- 
nishes this  vaccine  free  to  farmers.  The  results  of  this 
method  are  said  to  be  very  gratifying.1 

1  See  Recent  Annual  Reports,  Bureau  of  Animal  Industry,  U.  S. 
Department  of  Agriculture. 


IMMUNITY.  179 

In  the  human  disease,  bubonic  plague,  a  nearly  similar 
procedure  has  been  proposed  by  Haffkine.  To  protect 
against  plague,  cultures  of  plague  bacilli,  previously  steril- 
ized by  heat  and  carbolic  acid  are  injected.  (See  article  on 
Bubonic  Plague,  Part  IV.) 

Inoculation  Against  Rabies  or  Hydrophobia. — The  im- 
munity produced  in  this  case  probably  depends  upon  prin- 
ciples similar  to  those  underlying  the  examples  related  on 
the  preceding  pages.  But  this  question  cannot  be  regarded 
as  settled  until  the  organism  of  rabies  has  been  isolated  and 
cultivated.  Attempts  to  discover  this  organism  have,  as  yet, 
been  futile.  Pasteur  discovered  that  rabbits  are  susceptible 
to  rabies  when  portions  of  the  medulla  oblongata  of  a  dog 
which  has  died  of  the  disease  are  inserted  beneath  the  dura 
mater  of  the  rabbit.  Spinal  cords  taken  from  rabbits  thus 
injected  are  placed  in  a  desiccating  chamber.  Under  these 
circumstances  the  unknown  virus  undergoes  a  diminution 
in  virulence.  Emulsions  are  made  from  spinal  cords  desic- 
cated in  this  manner.  First  the  patient  is  injected  with  part 
of  an  emulsion  from  a  cord  which  has  been  desiccated  a 
longer  time,  and  in  which  the  virulence  of  the  poison  has 
been  much  reduced.  Injections  are  then  made  at  intervals 
from  cords  that  have  been  subjected  to  desiccation  for 
shorter  and  shorter  periods,  and  therefore  of  greater  and 
greater  virulence.  At  the  end  of  about  the  twenty-fifth  day 
the  patient  is  supposed  to  be  immune  from  rabies.  The 
period  of  incubation  in  rabies  is  longer  than  in  most  infec- 
tious diseases,  being  usually  one  to  two  months.  Al- 
though the  injections  take  place  after  he  has  been  bitten  by 
a  rabid  dog,  it  is  hoped  the  patient  may  be  rendered  immune 
before  the  period  of  incubation  has  ended.1 

Reports  of  cases  managed  according  to  this  method  have 
been  conflicting  in  the  past.  However,  there  seems  no  longer 

1  F.   Cabot,    "  The  Dilution   Method   of  Immunization   from  Rabies," 
Journal  Experimental  Medicine,  Vol.  IV.,  p.  181. 
16 


I  SO  MANUAL    OF    BACTERIOLOGY. 

to  be  any  question  as  to  its  efficiency.  Laboratories  where 
Pasteur's  method  of  treatment  is  used  now  exist  in  all  parts 
of  the  world.  According  to  statistics  collected  by  Ravenel, 
based  on  many  thousands  of  cases,  the  mortality  from  rabies 
in  those  so  treated  is  less  than  one  per  cent.1 

Antitoxins. — The  first  efforts  to  point  out  the  way  along 
which  antitoxins  might  be  secured  were  made  by  Salmon 
and  Smith  in  1886.  In  their  experiments  pigeons  were  in- 
jected with  filtrates  from  cultures  containing  the  products 
resulting  from  growth  of  the  hog-cholera  bacillus.  Such 
pigeons  were  found  to  be  immune  to  this  bacillus,  which 
is  pathogenic  to  ordinary  pigeons. 

As  was  stated  in  the  last  chapter,  bacterial  poisons  may  be 
of  twro  sorts.  In  one  group  the  poisons  occur  chiefly  within 
the  bodies  of  the  bacteria.  This  group  seems  to  contain  the 
majority  of  the  pathogenic  bacteria.  Methods  of  protection 
against  infections  caused  by  them  will  be  considered  here- 
after. In  the  other  group,  the  poisons  do  not,  for  the  most 
part,  remain  in  the  bodies  of  the  bacteria,  but  are  readily 
diffused  from  them  into  their  surroundings.  It  is  for  the 
bacteria  of  the  latter  group  that  antitoxins  have  been  made. 
Its  most  important  members  are  the  bacilli  of  diphtheria  and 
tetanus.  Their  poisons  may  be  found  in  the  culture-media 
in  which  they  have  grown.  The  principle  employed  in  pre- 
paring antitoxins  was  established  by  Behring.  The  bacilli 

lln  cases  of  dog-bite  there  is  often  some  doubt  as  to  whether  or  not 
the  dog  was  rabid.  The  dog  should  be  allowed  to  die  under  observa- 
tion. A  large  ganglion,  as  that  of  the  pneumogastric  nerve,  should  be 
removed  and  placed  in  absolute  alcohol  or  ten  per  cent,  fcrmaldehyde 
solution.  Microscopic  changes  have  been  observed  in  the  ganglia  which 
seem  to  be  quite  constant  in  rabies.  See  Ravenel  and  McCarthy,  Uni- 
versity of  Pennsylvania  Medical  Bulletin,  June,  1901  ;  also  editorial  in 
Philadelphia  Medical  Journal,  March  14,  1903.  The  diagnosis  may  be 
made  by  inoculating  a  rabbit  as  above  described,  but  the  results  are  not 
obtained  quickly  enough  to  be  of  value.  See  V.  A.  Moore,  ''Infectious 
Diseases  of  Animals." 


IMMUNITY.  l8l 

are  cultivated  in  bouillon.  The  cultures  are  freed  from  all 
living  bacilli  by  filtration.  The  liquid  filtrate  contains  the 
toxin.  This  filtrate  is  injected  into  healthy  animals,  usually 
horses,  in  increasing  doses.  Eventually  enormous  doses  of 
toxin  are  given,  and  the  animal  acquires  a  high  degree  of 
immunity.  The  blood  of  the  animal  is  withdrawn,  taking 
care  to  avoid  contamination.  The  serum  of  the  blood  is 
collected  and  constitutes  the  antitoxin.  Such  antitoxins 
have  produced  brilliant  results  in  the  treatment  of  diph- 
theria, and  have  given  partial  success  with  tetanus.  "  (See 
the  articles  on  the  bacteria  of  these  diseases.) 

Ehrlich  discovered  that  the  vegetable  toxins,  abrin  and 
ricin,  behave  in  a  manner  very  similar  to  soluble  bacterial 
poisons  when  injected  into  animals,  and  that  by  their  injec- 
tion an  immunity  for  the  same  poisons  may  be  secured. 
There  is  an  analogy  between  the  tolerance  acquired  in  this 
manner  from  bacterial  and  other  toxins  and  that  which  vic- 
tims of  the  morphine  and  cocaine  habits  have  for  immense 
doses  of  those  drugs.  Ehrlich  also  found  that  the  milk  of 
animals  which  had  been  immunized  against  abrin  and  ricin 
might  confer  immunity  upon  young  animals.  In  most  cases 
we  look  to  the  blood-serum  for  the  immunizing  agent. 

Active  and  Passive  Immunity. — The  kind  of  immunity 
which  results  from  the  injection  of  substances  from  im- 
munized animals  is  called  "  passive  immunity."  Diphtheria 
and  tetanus  antitoxins  produce  passive  immunity.  "  Active 
immunity  "  may  be  brought  about  in  several  ways : 

(i)  By  an  ordinary  attack  of  an  infectious  disease;  (2) 
by  an  attack  excited  artificially  through  inoculation  with 
small  doses  of  virulent  cultures,  or  (3)  large  doses  of  atten- 
uated cultures;  (4)  or  by  the  injection  of  bacterial  products 
(toxins)  freed  from  the  bacteria  themselves.  Pasteur's 
methods  of  protective  inoculation  for  anthrax,  etc.,  and 
Haffkine's  injections  for  bubonic  plague  produce  active 


1 82  MANUAL    OF    BACTERIOLOGY. 

immunity.  Active  immunity  is  usually  more  enduring  than 
passive  immunity.  Passive  immunity,  established  through 
the  direct  introduction  of  antitoxins,  may  be  brought  about 
more  quickly  than  would  be  possible  for  an  active  immunity. 

THEORIES  OF  IMMUNITY. 

Phagocytosis.1 — Metchnikoff  described  under  the  name 
"  phagocytosis  "  a  phenomenon  which,  he  maintained,  could 
explain  immunity  and  recovery  from  bacterial  invasion. 
This  theory  is  based  on  the  well-known  fact  that  certain  cells 
of  the  body  have  the  power  of  surrounding  and  ingesting 
foreign  substances.  The  cells  in  question  are  chiefly  poly- 
nuclear  leucocytes,  but  to  some  extent  other  leucocytes,  and 
enclothelial  and  other  cells.  There  are  many  examples  of 
this  process.  The  leucocytes  of  the  lungs  constantly  take  up 
small  bits  of  carbon  inhaled  with  the  air.  Particles  of  car- 
mine injected  into  the  tissues  will  later  be  found  within 
leucocytes.  After  a  hemorrhage,  phagocytic  cells  may  be 
found  containing  red  blood-corpuscles  or  particles  of  blood 
pigment.  The  presumption  is  that  phagocytic  cells  serve  to 
remove  irritating  and  foreign  bodies  to  less  sensitive  parts. 
Metchnikoff  showed  that  phagocytes  also  absorb  bits  of  de- 
generating or  useless  tissue.  Such  particles  disintegrate 
and  their  identity  is  lost.  They  are  digested  and  become  a 
part  of  the  protoplasm  of  the  phagocytes.  This  process  is 
seen  when  the  tail  of  the  tadpole  shortens.  The  superfluous 
part  is  absorbed,  at  least  in  part,  by  phagocytic  leucocytes. 
MetchnikofFs  observations  were  made  largely  on  the  in- 
vertebrates, whose  transparent  bodies  may  be  studied  while 
living.  One  illustration  was  furnished  by  a  small  crustacean 
(Daphnia  or  water-flea),  which  was  often  infected  with  a 
fungus.  Some  infected  individuals  died,  others  recovered. 
Metchnikoff  found  that  the  cells  of  the  fungus  might  be 

1  Greek,  (bayelv,  to  eat ;  nvroq ,  a  cell. 


IMMUNITY.  183 

ingested  and  destroyed  by  the  leucocytes  of  the  Daphnia. 
He  described  the  history  of  this  disease  as  a  contest  between 
the  parasitic  cells  and  the  phagocytes,  in  which  either  might 
succeed.  Similarly,  when  anthrax  bacilli  were  introduced 
into  frogs,  which  are  immune  from  anthrax,  the  bacilli  were 
ingested  by  the  frog's  leucocytes.  MetchnikofF  contended 
that  this  function  of  leucocytes  and  other  phagocytic  cells 
constituted  the  principal  defence  of  the  body  against  bac- 
teria. 

Other  investigators  also  have  seen  bacteria  enclosed 
within  the  bodies  of  leucocytes.  It  has  been  urged  by  some 
that  the  bacteria  are  already  dead  when  the  leucocytes  de- 
vour them.  In  some  cases,  as  with  the  gonococcus  which  is 
commonly  found  enclosed  within  leucocytes,  it  is  possible 
that  the  bacteria  retain  their  full  vigor  after  being  ingested. 

It  is  well  known  that  a  suppurating  part  contains  large 
numbers  of  leucocytes,  and  one  of  the  most  characteristic 
events  in  the  inflammatory  process  is  the  migration  of 
leucocytes  to  the  point  of  irritation.  This  indicates  a  posi- 
tive chemotaxis  for  leucocytes  on  the  part  of  substances  in 
the  inflamed  area.  Metchnikoff  believed  that  the  function 
of  these  leucocytes  is  to  destroy  the  bacteria  and  to  arrest 
their  further  progress.  On  this  theory  bacteria  have  often 
been  likened  to  an  invading  army  and  the  leucocytes  or 
phagocytes  to  a  force  designed  to  repel  their  attacks. 

It  is  certain  that  in  some  infectious  diseases  the  number 
of  leucocytes,  chiefly  of  the  polynuclear  neutrophilic  variety, 
in  the  circulating  blood  is  increased  (leucocytosis).  This  is 
the  case  usually  in  lobar  pneumonia  and  acute  suppurative 
infections.  In  other  infectious  diseases  there  is  no  leucocy- 
tosis ;  for  example,  tuberculosis,  typhoid  fever  and  malaria. 
It  is  interesting  to  observe  that  in  trichinosis,  and  more 
rarely  in  infection  with  other  animal  parasites,  the  eosino- 

1  Metchnikoff,  "  Comparative  Pathology  of  Inflammation,"  trans., 
Starling,  1893. 


184  MANUAL    OF    BACTERIOLOGY. 

philic  leucocytes  become  much  more  numerous  in  the  blood 
than  normally.  There  is  every  reason  for  believing  that  the 
leucocytes  and  other  body  cells  play  an  important  part  in 
combating  bacteria.  However,  the  doctrine  of  phagocytosis, 
as  primarily  stated,  is  insufficient  to  account  for  many  facts. 
Metchnikoff  himself  has  modified  his  original  views,  to  con- 
form with  more  recent  studies. 

Ehrlich's  Side-Chain  Theory  of  Immunity.1 — This  the- 
ory was  first  proposed  to  explain  the  resistance  of  the 
body  to  soluble  bacterial  poisons,  as  those  of  diphtheria  and 
tetanus.  In  these  cases  what  is  known  as  antitoxic  immunity 
is  produced.  With  the  infections  in  which  the  poisons  occur 
in  the  bodies  of  the  bacteria  the  protective  mechanism  is 
different,  giving  rise  to  the  term  bacteriolytic  immunity. 

Antitoxic  Immunity. — Ehrlich  first  endeavored  to  ex- 
plain from  a  chemical  standpoint  the  action  of  toxins  on 
cellular  protoplasm  and  the  formation  of  antitoxins.  To 
begin  with,  the  molecules  of  the  protoplasm  are  to  be  re- 
garded as  being  endowed  with  chemical  groups,  present  in 
the  form  of  lateral  appendages  to  the  molecule,  called  side- 
chains.  They  can  be  illustrated  by  the  analogies  presented 
by  the  graphically  written  formulae  of  some  complex  mole- 
cules. It  is  necessary  to  conceive  of  molecules  made  of  an 
immense  number  of  atoms,  and  bristling  with  projecting 
side-chains.  The  function  of  the  side-chains  is  to  become 
attached  to  other  organic  molecules  with  which  thev  have 
affinities.  In  this  manner  they  aid  in  absorbing  the  sub- 
stances essential  for  the  nutrition  of  the  protoplasm  of  cells. 

1The  literature  of  this  subject  is  very  extensive.  An  exhaustive 
review  is  that  by  L.  Aschoff,  "  Ehrlich's  Seitenkettentheorie,"  ZeitscJmft 
f.  aUgcmcinc  Physiologic,  1902. 

The  following  are  also  of  a  general  character:  H.  C.  Ernst,  "  Modern 
Theories  of  ^pcternl  Immunity,"  1903;  Prudden,  Medical  Record, 
February  i  1.  1003;  RilrlrV.  Journal  of  Hygiene,  Vol.  II.,  1902;  Bergey, 
American  Medicine,  October  n,  1902. 


IMMUNITY.  155 

The  side-chains  are  therefore  preferably  called  "  receptors" 
The  numerous  receptors  which  a  molecule  has  are  of  many 
kinds,  with  affinities  for  other  molecules  of  different  kinds. 
Each  kind  of  receptor  will  then  have  an  affinity  for  a  mole- 
cule of  a  particular  kind,  which  it  may  be  said  to  "  fit,"  as  a 
key  fits  in  a  lock,  although  this  expression  must  not  be  taken 
in  a  literal  sense.  A  receptor  to  which  tetanus  toxin  might 
become  attached  would  not  "  fit  "  diphtheria  toxin.  In  order 
that  toxins  may  be  able  to  combine  with  the  receptors  their 
structure  must  be  nearly  like  that  of  the  food  molecules 
which  the  receptors  are  adapted  to  receive. 

FIG.  48. 


\— Antitoxin 
Body  cell       >          Antitoxin 

Diagram  to  Illustrate  Side-chain  Theory   (modified  from  Aschoff). 

Secondly,  soluble  toxins  are  to  be  looked  upon  as  definite 
chemical  bodies  excreted  by  bacteria,  and  containing  two 
essential  groups.  One  group  is  the  haptophorc,  by  means  of 
which  the  toxin  may  be  linked  with  the  receptors  of  the 
molecules  of  the  cell.  The  other  group  is  the  ioxophore, 
which  is  capable  of  destroying  the  protoplasmic  molecule, 
after  being  attached  to  the  receptor  of  the  latter  by  the 
haptophore. 

These  relations  may  be  represented  schematically  though 
in  a  very  crude  manner.  In  Fig.  48  a  portion  of  a  cell  is 
shown,  with  receptors.  Molecules  of  toxin,  with  hapto- 
phores  and  toxophores  are  near  by  or  attached.  At  the 


1 86  MANUAL    OF    BACTERIOLOGY. 

right  we  see  a  free  receptor  (antitoxin)  and  one  which  has 
united  with  toxin  to  neutralize  it. 


II 

/\ 

/\ 

COOH 

/\ 

\H 

/  \ 
H/        \H 

/  \ 

1  1/        \OH 

C6 

. 

C6 

/H 

H\ 

H 

Hlrr 
\                 /•** 

\        / 

\       / 

\        / 

\/ 
II 

Y 

Y 

Benzol. 

Phenol. 

Salicylic  Acid. 

The  chemical  nature  of  this  conception  may  be  illustrated 
by  recalling  the  formula  of  a  molecule  of  benzol.  Here  one 
or  more  H  atoms  may  be  replaced  with  equivalent  groups 
making  phenol,  salicylic  acid  and  other  derivatives. 

As  the  side-chains  or  receptors  of  the  protoplasm  are 
essential  to  its  existence,  their  combination  with  the  toxin, 
through  its  haptophore,  might  result  in  destruction  of  the 
molecule.  But  if  the  damage  be  not  too  serious,  the  proto- 
plasm may  be  stimulated  to  produce  numerous  similar  side- 
chain  groups.  Not  all  of  these  are  necessary  for  the  per- 
formance of  its  functions,  and  the  superfluous  ones  are 
thrown  off  into  the  surrounding  serum.  It  is  well  known 
that  many  cells  of  the  body  exhibit  analogous  heightened 
activities  under  stimulating  influences.  Such  free  side- 
chains  or  receptors  may  combine  with  the  haptophorous 
groups  of  the  toxin,  when  it  will  no  longer  be  able  to  com- 
bine with  the  protoplasm.  Thus  they  act  as  a  kind  of  buffer 
in  protecting  the  protoplasm  from  the  attacks  of  the  toxins. 
They  constitute  the  antitoxic  part  of  the  serum. 

Numerous  experiments  have  been  made  which  illustrate 
the  probable  chemical  nature  of  antitoxic  action.  A  fatal 
dose  of  diphtheria  or  tetanus  toxin  may  be  neutralized  out- 
side of  the  body  by  mixing  it  with  its  appropriate  antitoxin. 
Injection  of  the  mixture  shows  it  to  be  innocuous  to  animals. 

The  manner  in  which  toxins  combine  with  protoplasm  has 


IMMUNITY.  187 

been  shown  in  the  case  of  tetanus  toxin.  The  filtrate  from 
cultures  of  tetanus  bacilli  will  kill  guinea-pigs,  presumably 
by  damage  to  the  central  nervous  system.  The  same  filtrate 
rubbed  up  with  brain  or  spinal  cord  has  been  found  to  have 
lost  its  toxic  proporties.  It  may  be  assumed  that  the  poison 
has  combined  with  the  protoplasm  of  the  cells. 

Bacteriolysis. — Although  the  hypothesis  of  Ehrlich  just 
stated  may  seem  very  complicated,  more  recent  studies  along 
similar  lines  have  led  to  conceptions  so  intricate,  that  they 
can  be  followed  only  with  the  greatest  difficulty.  The 
nomenclature  of  this  subject  has  also  become  extremely 
involved,  as  different  words  have  often  been  coined  to  con- 
vey a  similar  idea.  In  other  cases  a  single  word  has  been 
used  in  different  senses. 

The  problems  we  have  still  to  consider  relate  to  the  bac- 
teria which  do  not  produce  soluble  toxins.  In  infections 
with  these  organisms,  attempts  to  make  protective  or  cura- 
tive substances  analogous  to  antitoxins  have  met  with  little 
success.  The  outlook  for  better  results  in  the  future  is 
promising. 

The  agencies  which  lead  to  the  destruction  of  bacteria 
during  the  progress  of  an  infectious  disease  have  been 
studied  chiefly  in  experimental  infections  in  the  lower  ani- 
mals. There  are  numerous  bacteria,  such  as  the  spirillum 
of  cholera,  bacillus  of  typhoid  and  bacillus  coli  communis, 
injection  of  which  may  produce  a  fatal  septicemia  in  ani- 
mals. It  also  usually  is  possible  to  render  animals 
immune  to  them  by  injections  of  the  dead  bacteria,  or 
small  doses  of  living  bacteria,  or  the  two  in  succession. 
When  an  animal  has  been  immunized  in  this  manner,  in- 
jection of  living  cultures  of  the  same  kind  is  followed  by 
destruction  of  the  bacteria  injected.  This  phenomenon 
appears  to  depend  on  the  combined  action  of  two  substances. 
Neither  of  these  is  effective  without  the  other.  The  actual 


1 88  MANUAL    OF    BACTERIOLOGY. 

destruction  of  bacteria  (bacteriolysis)  is  performed  by  the 
complement,  an  unstable  body,  destroyed  at  quite  low  tem- 
peratures (about  60°  C.).  The  union  of  the  complement 
with  the  bacterial  cell  requires  the  presence  of  an  inter- 
mediary body,  which  is  more  stable,  and  more  resistant  to 
heat. 

Some  of  the  synonyms  for  these  terms  are : 

Complement,  Intermediary  Body, 

Addiment,  Amboceptor, 

Cytase,  Immune  Body, 

Alexin,  Substance  Sensibilitrice. 

The  nature  of  complements  and  intermediary  bodies  has 
in  a  large  measure  been  worked  out  by  the  study  of  im- 
munity to  substances  not  directly  related  with  bacteriology. 
Among  these  the  blood1  of  various  animals  and  snake 
venom2  are  notable  examples. 

The  substances  developed  or  increased  by  immunization 
are  the  intermediary  bodies,  which  have,  therefore,  also  been 
called  immune  bodies.  Their  ultimate  source  must  have 
been  in  the  cells  of  the  host.  They  may  be  supposed  to  have 
been  receptors  originally,  which  were  cast  off,  after  the 
manner  of  antitoxins.  They  are  specific  for  the  particular 
kind  of  organism  used  in  developing  immunity.  Whether 
or  not  various  kinds  of  complements  exist  is  still  in  doubt. 

Metchnikoff  holds  that  while  intermediary  bodies  may 
occur  in  blood-serum,  the  complement  exists  within  leuco- 
cytes and  other  forms  of  cells.  Union  of  intermediary 
bodies  with  the  molecules  of  a  bacterial  cell,  permits  de- 
struction of  the  bacteria  by  the  complement  in  the  phagocyte. 

1  Prtidden,  Medical  Record,  February  14,  1903. 

2  Flexner   and   Noguchi,    "  Snake   Venom    in   Relation   to   Hemolysis, 
etc.,"  Journal  Experimental  M'edicine.  Vol.  VI.,   1902,  p.  277.     Univer- 
sity of  Pennsylvania  Medical  Bulletin,  November;  1902. 


IMMUNITY.  189 

Disintegration  of  leucocytes  may  give  rise  to  the  presence 
of  complement  in  the  serum. 

An  example  of  the  destruction  of  bacteria  in  immunized 
animals  is  seen  in  an  experiment  performed  by  Pfeiffer.  A 
guinea-pig  is  given  repeated  injections  of  the  spirilla  of 
cholera,  which  have  been  killed  by  chloroform  or  heat. 
When  living  spirilla  are  introduced  into  the  peritoneal 
cavity  of  such  an  immunized  guinea-pig  they  rapidly 
undergo  disintegration.  Since  no  such  disintegration  takes 
place  when  other  bacteria  than  the  spirilla  of  cholera  are  in- 
jected into  the  animal  made  immune  from  this  organism,  it 
has  been  suggested  by  Pfeiffer  that  this  reaction  could  be 
made  use  of  in  the  diagnosis  of  that  disease.  If  the  serum 
of  the  immune  animal  be  introduced  along  with  the  cholera 
spirilla  into  the  peritoneal  cavity  of  an  animal  not  immune, 
the  same  disintegration  takes  place.  Furthermore  the  serum 
of  the  immune  animal  may  be  heated  to  70°  C.  and  will  still 
cause  disintegration  of  the  organisms  in  the  peritoneum 
of  a  non-immune  guinea-pig.  The  heated  serum  alone  is 
found  inactive.  The  explanation  of  this  phenomenon  ap- 
pears to  be  that  the  serum  of  the  normal  animal  contains 
the  complement  only.  The  serum  of  the  immunized  animal 
has  developed  in  it  the  intermediary  or  immune  body,  besides 
the  normal  complement.  The  immune  body  resists  heat, 
while  the  complement  is  destroyed  by  heat.  The  previously 
heated  serum  from  the  immunized  animal,  mixed  with 
organisms,  finds  the  necessary  complement  in  the  normal 
animal.  The  organisms  then  become  disintegrated. 

Such  disintegration  of  bacteria  is  called  bacteriolysis. 
The  substances  which  effect  it  are  called  lysins.  It  is  prob- 
able that  the  development  of  lysins  is  one  of  the  most  im- 
portant factors  in  checking  infections  and  in  promoting 
recovery.  The  preparation  of  specific  bacteriolytic  blood- 
serum  from  immunized  animals  has  been  attempted  for  the 


190  MANUAL    OF    BACTERIOLOGY. 

treatment  of  various  diseases.  The  partial  or  complete  fail- 
ure of  such  efforts  may  be  ascribed  to  the  fact  that  the 
serum  prepared  does  not  contain  both  the  complement  and 
the  intermediary  or  immune  body  in  proper  kinds  and  pro- 
portions to  be  effective.  There  is  likely  to  be  a  deficiency 
of  complement.  Investigators  are  at  present  very  hopeful 
of  being  able  to  make  up  for  this  deficiency.  The  outlook 
is  encouraging  at  the  present  time  particularly  with  regard 
to  typhoid  fever  and  dysentery. 

Welch's  Hypothesis. — When  bacteria  do  not  form  sol- 
uble toxins,  we  are  obliged  to  suppose  that  large  numbers 
of  them  must  disintegrate  in  the  body  of  the  host.  Thus 
their  intracellular  toxins  may  be  liberated  and  produce  the 
symptoms  of  intoxication  manifested  in  infections  of  this 
character.  Welch  has  also  suggested  that  production  of 
toxins  may  occur  in  infection  of  the  living  body  even  when 
test-tube  cultures  of  the  same  bacteria  contain  no  soluble 
toxins  or  only  small  amounts.  Bacteria  growing  as  para- 
sites in  the  living  body  may  adapt  themselves  to  the  condi- 
tions found  there.  The  existence  of  the  bacteria  requires 
them  to  have  some  means  of  combating  the  substances 
elaborated  by  the  host  for  his  protection.  Bacteria  may 
also  be  able  to  form  similar  substances  for  their  own  protec- 
tion. Such  substances,  from  the  standpoint  of  the  host, 
would  constitute  toxins.1 

Nuttall  made  the  important  discovery  that  the  serum  of 
the  blood  deprived  of  all  cells  possesses  the  power  of  destroy- 
ing pathogenic  bacteria. 

The  ingredients  of  the  blood-serum  that  exert  the  bacteri- 
cidal influence  have  been  named  alexins,2  they  have  also 
been  called  defensive  proteids.  The  nature  of  these  sub- 
stances has  not  been  determined  with  certainty.  Vaughan 

1  Welch,  Bulletin  Johns  Hopkins  Hospital,  December,   1902. 

2  This  word  has  unfortunately  been  used  partly  in  another  sense,  sec 
page  1 88. 


IMMUNITY. 

has  shown  that  they  may  be  in  part  nucleins.  He  has  found 
that  blood-serum  contains  nuclein  and  that  nuclein  has 
germicidal  power.  Such  substances  apparently  serve  as 
safeguards  to  the  body  against  all  kinds  of  bacterial  in- 
vasion. They  have  not  necessarily  a  specific  action  as 
regards  any  particular  kind  of  infection. 

It  now  appears  that  the  bactericidal  power  of  blood-serum 
also  depends  in  a  large  measure  upon  the  joint  operation  of 
complements  and  intermediary  bodies. 

The  amount  of  intermediary  body  appears  to  be  more 
constant  than  that  of  the  complement.  Flexner1  found 
that  the  blood-serum  from  cases  of  chronic  diseases  had 
diminished  bactericidal  power  for  the  staphylococcus  pyo- 
genes  aureus.  His  observations  were  regarded  as  explain- 
ing the  liability  of  these  patients  to  terminal  infections. 
(See  page  169.)  Longcope2  has  since  shown  that  in  such 
affections  as  chronic  nephritis,  cirrhosis  of  the  liver  and 
diabetes  the  complement,  active  in  destroying  bacteria,  is 
diminished.  Abbott  and  Bergey3  proved  that  the  comple- 
ment active  in  disintegrating  the  blood-corpuscles  of  another 
species  is  reduced  in  quantity  by  alcoholic  poisoning.  Their 
conclusions  are  important  taken  in  connection  with  Abbott's 
previous  work  on  the  effect  of  alcohol  in  lowering  the  re- 
sisting power  to  infection  (see  page  166). 

Agglutinins.4 — It  has  been  shown  that  the  blood-serum 
of  patients  having  typhoid  fever  contains  a  substance  which, 
when  mixed  with  living  typhoid  bacilli,  causes  them  to 
gather  into  groups  or  clumps  and  at  the  same  time  to  lose 
their  motility.  In  the  great  majority  of  cases  no  such 
clumping  occurs  when  the  blood  of  typhoid  cases  is  mixed 

1  Journal  of  Experimental  Medicine,  Vol.  L,  1896,  p.  21. 

2  University  of  Pennsylvania  Medical  Bulletin,  November,  1902,  p.  331. 

3  University  of  Pennsylvania  Medical  Bulletin,  Aug.-Sept.,  1902,  p.  186. 

4  Sailer,    University    of   Pennsylvania,   Medical   Bulletin,    Aug.-Sept., 
1902. 


IQ2  MANUAL    OF    BACTERIOLOGY. 

with  other  bacteria  than  the  bacilli  of  typhoid  fever.  The 
nature  of  this  agglutinating  substance,  as  it  is  called,  is  not 
known  nor  is  its  significance  understood.  It  has  been 
applied  to  the  diagnosis  of  typhoid  fever,  where  it  is  called 
the  "  serum-reaction,"  and  will  be  discussed  in  connection 
with  the  bacilli  of  typhoid  fever.  Similar  agglutinating 
bodies  form  in  many  other  infections.  Bacteria  that  are  not 
motile  may  nevertheless  be  agglutinated.  Among  the  in- 
fections where  such  a  reaction  occurs,  the  following  are 
noteworthy — with  the  cholera  spirillum,  bacillus  pyocya- 
neus,  bacillus  proteus,  bacillus  coli  communis,  micrococcus 
melitensis,  bacillus  of  glanders,  bacillus  tuberculosis,  diplo- 
coccus  of  pneumonia,  bacillus  of  bubonic  plague,  and  the 
bacillus  of  dysentery  (Shiga).  The  protozoon,  Try- 
panosoma  (see  appendix),  is  said  to  become  agglutinated 
in  the  blood  of  infected  rats.  Not  all  of  these  have  been 
studied  sufficiently  to  be  available  for  diagnostic  purposes. 
It  has  not  yet  been  shown  that  agglutinins  are  concerned  in 
producing  immunity  or  recovery  from  infectious  diseases. 
These  substances  are  relatively  resistant  to  heat,  not  being 
destroyed  by  temperatures  below  70°  C. 

Under  conditions  similar  to  those  under  which  agglutina- 
tion occurs,  bacteria  have  been  observed  to  form  in  long 
filaments.  This  is  the  so-called  "  thread-reaction."  Its  sig- 
nificance is  uncertain. 

Precipitins. —After  repeated  injections  of  albumins  for- 
eign to  it,  an  animal's  fluids  undergo  still  another  form  of 
adaptation.  Substances  are  now  developed  in  the  blood- 
serum,  which  cause  precipitates  to  form  where  it  is  mixed 
with  the  foreign  albumin.1  Thus  a  rabbit  may  be  immu- 

1  These  reactions  have  been  most  fully  studied  in  connection  with  the 
precipitation  of  blood-serum  of  a  particular  animal  by  the  serum  of 
another  species  which  has  been  immunized  to  the  serum  of  the  first 
animal.  The  principle  is  of  some  importance  in  medico-legal  work 
for  the  identification  of  human  blood,  as  in  stains  on  clothing.  See 
Nuttall,  Journal  of  Hygiene,  Vol.  I.,  1901. 


193 


IMMUNITY. 


nized  or  adapted  to  hen's  egg-albumen.  The  rabbit's  serum 
will  then  precipitate  hen's  egg-albumen,  but  no  other  form 
of  albumin.  It  may,  however,  imperfectly  precipitate  al- 
bumen from  the  egg  of  a  species  closely  allied  to  the  henr 
It  has  not  been  shown  that  this  property  plays  any  important 
part  in  bacteriology,  though  that  is  not  improbable. 


194  MANUAL    OF    BACTERIOLOGY. 


CHAPTER   VIII. 

DISINFECTANTS   AND  ANTISEPTICS.1 

A  disinfectant  or  germicide  is  a  substance  capable  of 
killing  bacteria.  The  latter  term  is  of  more  recent  develop- 
ment than  the  former,  and  apparently  needed  on  account 
of  the  loose  application  of  the  term  disinfectant. 

An  antiseptic  is  a  substance  capable  of  preventing  the 
growth  and  reproduction  of  bacteria.  It  differs  from  a 
disinfectant  or  germicide  in  that  it  simply  prevents  devel- 
opment without  actually  killing. 

A  deodorizer  is  a  substance  capable  of  so  changing  a 
noxious  odor  that  it  is  less  unpleasant  to  the  sense  of  smell. 
At  the  present  time  the  term  is  usually  and  properly  re- 
stricted to  those  substances  which,  without  disinfectant  ac- 
tion, simply  replace  or  destroy  an  odor. 

TESTING  ANTISEPTICS  AND  DISINFECTANTS. 

The  determination  of  the  antiseptic  value  of  a  material 
is  a  comparatively  simple  matter.  A  virulent  culture  of 
the  organism  used  as  a  test  is  inoculated  into  sterile  bouil- 
lon containing  a  known  quantity  of  the  antiseptic.  The 
process  is  repeated  with  varying  strengths  of  the  material 
until  the  smallest  quantity  of  it  capable  of  preventing 
growth  is  determined.  This  dilution  may  be  considered 
the  antiseptic  value  of  the  material  in  question  for  the  organ- 
ism used,  under  the  conditions  of  the  test. 

1  By    Thomas    B.    Carpenter,     M.D.,    Assistant    City    Bacteriologist, 
Buffalo,  N.  Y. 


DISINFECTANTS    AND    ANTISEPTICS.  IQ5 

Determination  of  the  disinfectant  power  of  a  substance 
is  a  problem  of  much  greater  magnitude,  and  the  method 
used  must  be  altered  more  or  less  to  suit  the  properties  of 
the  substance  tested.  It  is  obvious  that  some  of  the  sub- 
stance tested  remains  in  contact  with  the  organisms  in  the 
method  given  for  determining  the  antiseptic  value,  and 
that  we  do  not  know  whether  the  bacteria  are  alive  and 
merely  inhibited  in  growth,  or  actually  killed. 

Sternbergs  Method. — To  a  measured  quantity  of  a 
virulent  bouillon-culture  of  the  test-organism  is  added  a 
known  quantity  of  the  substance  to  be  tested.  After  vary- 
ing lengths  of  time  inoculations  are  made  from  this  mixture 
into  culture-media,  preferably  bouillon,  and  growth  watched 
for  under  suitable  conditions  as  to  temperature  and  the  like. 
The  shortest  exposure  of  the  test-organism  to  the  smallest 
quantity  of  the  substance  is  taken  as  the  germicidal  value 
of  that  substance  for  the  particular  organism  used. 

Koch's  Method. — Usually  employed  for  spore-bearing 
bacteria  like  the  bacillus  of  anthrax.  The  hay  bacillus  is 
convenient  to  use  when  experiments  are  being  made  by 
large  classes  of  students.  Small  pieces  of  sterile  silk  or 
cotton  thread  are  soaked  for  some  hours  in  a  bouillon-cul- 
ture of  the  test-organisms.  They  are  removed,  partially 
dried,  and  then  placed  in  a  solution  of  known  strength  of 
the  substance  being  tested,  and  exposed  for  a  definite 
length  of  time.  The  thread  is  removed  from  the  solution, 
•  washed  carefully  in  sterile  water,  planted  in  bouillon,  and 
growth  is  watched  for.  As  in  other  methods,  the  greatest 
dilution  of  the  germicide  that  will  kill  the  test-organism  in 
the  shortest  time  is  taken  as  the  germicidal  value  of  that 
substance  for  the  organism  used. 

Hill's  Method. — The  test  organism  is  dried  upon  the  end 
of  a  sterile  glass  rod  contained  in  a  sterile  test-tube,  the  end 
of  the  rod  projecting  through  a  cotton  plug.  The  end  of 
17 


196  MANUAL    OF    BACTERIOLOGY. 

the  rod  is  inoculated  with  a  small  amount  of  a  fluid  culture 
of  the  test  organism  and  allowed  to  dry.  It  is  then  ready 
to  test  by  exposure  to  any  disinfectant,  either  liquid  or 
gaseous. 

All  of  these  methods  are  open  to  serious  sources  of  error, 
particularly  in  the  testing  of  powerful  germicides.  In 
Sternberg's  method,  small  quantities  of  the  substances 
tested  may  be  carried  over  with  the  organisms,  and,  if  a 
powerful  germicide,  in  sufficient  amount  to  prevent  growth, 
and  thus  give  erroneous  results.  In  Koch's  or  Hill's 
method  this  factor  may  be  partially  obviated  by  washing  in 
sterile  water  after  exposure  to  the  germicide.  This  does  not 
remove  another  source  of  error,  namely,  the  chemical  action 
that  may  take  place  between  the  substance  and  the  proto- 
plasmic contents  of  the  bacterial  cell.  This  action  may  ex- 
tend deeply  enough  to  restrain  the  growth  of  an  organism 
for  a  very  long  time  without  actually  killing  it.  When 
placed  under  suitable  conditions,  such  union  may  be  broken 
up  and  the  organism  regain  its  power  to  develop.  It  has 
been  suggested  that,  to  remove  errors  in  the  above  methods, 
test-cultures  containing  bacteria  supposed  to  be  killed  by 
the  smallest  quantity  of  germicide  be  inoculated  into  sus- 
ceptible animals;  but  Sternberg's  experiments  in  this  direc- 
tion have  shown  that  bacteria  may  become  so  altered  in 
virulence  by  the  action  of  germicides  insufficient  to  kill,  that 
animal  inoculation  experiments  are  worthless. 

Geppert  suggested  a  valuable  modification  of  these 
methods  while  determining  the  germicidal  value  of  bichlo- 
ride of  mercury.  After  exposing  his  test-organism  to 
bichloride  of  mercury,  and  before  inoculating  into  bouillon 
to  determine  death  of  the  organism,  he  treated  with  a  dilute 
ammonium  sulphide  solution,  thus  effectually  neutralizing 
any  mercury-salt  remaining. 

Sedgwick  developed  this  method  still  further,  and  to  him 


DISINFECTANTS    AND    ANTISEPTICS.  197 

we  are  indebted  for  demonstrating  its  accuracy  and  "practi- 
cability. 

Method. — To  15  c.c.  of  sterile  water  in  a  60  c.c.  Erlen- 
meyer  flask  add  2  c.c.  of  a  virulent  culture  of  the  test- 
organism.  Then  add  a  solution  of  the  substance  under  in- 
vestigation in  the  proportion  necessary  to  give  the  dilution 
wished.  Mix  thoroughly,  and  allow  this  "  action-flask  "  to 
stand  as  long  as  it  is  desired  to  have  the  germicide  in  con- 
tact with  the  test-organism  (action-period).  Transfer  0.5 
c.c.  from  the  action-flask  to  a  flask  containing  200  c.c.  of  a 
solution  of  some  chemical  capable  of  decomposing  the  sub- 
stance being  tested  with  the  formation  of  inert  or  insoluble 
compounds.  In  this  "  inhibition-flask  "  the  strength  of  the 
solution  should  be  such  that  molecular  proportions  of  the 
chemical  are  present  in  sufficient  quantity  to  combine  with 
all  the  germicide  carried  over.  The  inhibition-flask  is 
shaken  for  30  seconds,  and  i  c.c.  transferred  from  it  to  100 
c.c.  of  sterile  water  in  another,  the  "  dilution-flask."  After 
two  minutes,  three  agar  tubes  are  inoculated  with  i  c.c.  each 
from  the  dilution-flask,  plated,  and  growth  watched  for. 

Control-experiments  should  be  performed  to  determine 
that  the  dilution  of  the  test-culture  is  not  too  great  when 
carried  through  the  three  flasks.  It  likewise  should  be  de- 
termined that  the  inhibiting  chemical  has  no  effect  on  the 
bacteria. 

What  the  inhibiting  chemical  shall  be  must  be  deter- 
mined for  each  individual  case.  For  salts  of  the  heavy 
metals  ammonium  sulphide  answers  well ;  for  mercury  salts, 
stannous  chloride  may  be  used;  for  formaldehyde,  ammo- 
nium hydrate;  for  carbolic  acid,  sodium  sulphate. 

The  testing  of  gaseous  disinfectants,  such  as  sulphur 
dioxide  and  formaldehyde,  must  be  conducted  under  condi- 
tions as  nearly  parallel  to  actual  practice  as  possible.  The 
test-organisms  may  be  exposed  on  threads  or  glass  rods,  and 


198  MANUAL    OF    BACTERIOLOGY. 

acted  upon  by  a  known  volume  strength  of  disinfectant  for 
a  known  length  of  time.  Subsequent  treatment  of  the 
organisms  with  a  suitable  inhibitor  is  necessary  when  pos- 
sible, and  should  growth  occur  in  the  cultures  following,  the 
test-organism  should  be  recognized  in  order  that  possible 
contamination  by  extraneous  organisms  may  be  excluded. 

In  determining  the  value  of  germicides  for  sterilizing 
ligatures,  the  students  can  apply  methods  based  on  the  fore- 
going principles.  Great  care  and  ingenuity  are  necessary 
to  arrive  at  correct  conclusions,  particularly  in  the  case  of 
animal  tendons.  In  many  instances  quite  stable  compounds 
are  formed  between  tendon  and  germicide,  and  living  or- 
ganisms may  be  so  imbedded  in  such  a  substance  that  sub- 
sequent growth  in  a  test-culture  is  impossible.  The  use  of 
a  suitable  inhibitor,  and,  prior  to  final  culture-tests,  a  pro- 
longed soaking  in  sterile  water,  will  promote  the  accuracy 
of  the  results. 

CHEMICAL  DISINFECTION. 1 

Heat  properly  applied  is  the  simplest  and  at  the  same 
time  the  surest  disinfectant  (see  Part  I.,  Chapter  II.)  ;  but 
for  many  purposes  it  cannot  be  used,  and  we  have  recourse 
to  those  chemicals  that  practice  and  investigation  have 
shown  to  be  of  value.  The  efficiency  of  chemical  disin- 
fectants as  ordinarily  used  is  over-rated.  An  immense 
number  of  substances  possess  germicidal  properties,  but  un- 
fortunately, the  majority  are  objectionable  in  that  they  are 
expensive,  intensely  poisonous,  or  so  corrosive  that  damage 
may  be  done  to  articles  of  value  with  which  they  may  come 
in  contact. 

In  the  following  pages  only  those  substances  which  are 
in  common  use  or  seem  to  be  of  special  value  will  be  con- 
sidered. 

1  For  fuller  details  on  this  subject  consult  Rosenau,  "Disinfection  and 
Disinfectants,"  1902. 


DISINFECTANTS    AND    ANTISEPTICS. 

Mercuric  Chloride  or  Bichloride  of  Mercury. — This 
substance  is  probably  more  commonly  used  than  any  other 
one  disinfectant.  In  the  strength  of  i-iooo  it  will  some- 
times kill  the  spores  of  anthrax  within  a  few  minutes  (see 
Bacillus  anthracis,  Part  IV.).  It  is  claimed  that  its  affinity 
for  albuminous  bodies,  and  the  readiness  with  which  it 
combines  with  such  substances,  detracts  from  its  value  for 
some  purposes.  On  the  other  hand,  many  observers  claim 
that  the  albuminous  combinations  formed  under  such  cir- 
cumstances are  soluble  in  an  excess  of  albuminous  fluid, 
and  that  its  value  as  a  germicide  is  not  affected  thereby. 
To  obviate  this  possible  difficulty  it  is  customary  in  prac- 
tice to  combine  the  bichloride  of  mercury  with  some  sub- 
stance that  will  prevent  the  precipitation  of  the  mercury 
salt  by  albumin.  For  this  purpose  5  parts  of  any  one  of 
the  following  substances  to  i  part  of  bichloride  of  mercury 
may  be  used — hydrochloric  acid,  tartaric  acid,  sodium 
chloride,  potassium  chloride  or  ammonium  chloride.  A 
very  practical  stock-solution  for  laboratory  purposes  has 
the  following  composition : 

Hydrochloric  acid  100  c.c. 

Bichloride  of  mercury  20  grams. 

5  c.c.  in  a  liter  of  water  makes  a  solution  of  about  i-iooo  strength. 

Mercuric  Iodide. — An  extremely  high  antiseptic  value 
has  been  placed  on  this  substance  by  Miquel,  who  claims 
that  the  most  resistant  spores  are  prevented  from  develop- 
ing in  a  culture-medium  containing  1-40,000.  In  combi- 
nation, as  potassio-mercuric  iodide,  it  has  been  used  in 
soaps  (McClintock)  with  very  favorable  results.  The  sub- 
stance is  not  extensively  employed,  and  further  investiga- 
tion is  necessary  to  determine  its  true  value. 

Attempts  are  being  made  to  manufacture  combinations 
of  mercury  and  other  powerful  metallic  germicides  with 
organic  acid  and  basic  bodies,  the  purpose  being  to  utilize 


2OO  MANUAL    OF    BACTERIOLOGY. 

the  metallic  base  in  greater  strength  without  injury  to  the 
living  tissues.  Such  compounds  are  exemplified  by  uicr- 
curol,  said  to  be  a  combination  of  mercury  with  nucleinic 
acid,  and  to  possess  active  germicidal  properties,  great 
penetrating  power  and  no  injurious  effect  on  living  tissue. 
It  is  also  said  to  have  a  particularly  destructive  action  upon 
the  gonococcus. 

Silver  Nitrate. — This  salt  probably  occupies  the  next 
position  to  the  bichloride  of  mercury  in  disinfectant  power. 
Behring  claims  it  to  be  superior  to  bichloride  of  mercury 
in  albuminous  fluids.  The  anthrax  bacillus  is  killed  by  a 
solution  of  1-20,000  after  two  hours'  exposure.  At  least 
forty-eight  hours'  exposure  to  a  i-i  0,000  solution  is  re- 
quired to  kill  the  spores  of  anthrax.  It  is  very  irritating, 
and  possesses  strong  affinities  for  chlorides,  forming  with 
them, insoluble  chloride  of  silver,  a  salt  without  germicidal 
value.  For  these  reasons  the  use  of  silver  nitrate  is  lim- 
ited. In  the  solutions  usually  employed  for  douching  the 
cavities  of  the  body,  the  available  silver  nitrate  is  imme- 
diately converted  into  the  insoluble  chloride,  and  little  if 
any  germicidal  action  takes  place.  To  this  fact  may  be 
ascribed  the  varying  clinical  results  reported. 

Many  semi-proprietary  silver  compounds  are  on  the  mar- 
ket, introduced  to  replace  the  nitrate  and  its  objection- 
able features.  The  most  important  are  argentamin,  ar- 
gonin,  protargol  and  argyrol,  all  organic  silver  combina- 
tions. They  do  not  combine  with  chlorides,  are  less  irritat- 
ing than  the  nitrate,  and,  not  coagulating  albumin,  they  pos- 
sess greater  penetrating  power.  Clinical  reports  and  in- 
vestigations have  been  so  contradictory  thus  far  that  their 
value  cannot  be  readily  estimated. 

Carbolic  Acid. — One  of  the  most  important  and  most 
widely-used  disinfectants.  It  is  usually  employed  in 
strengths  of  from  i  to  5  per  cent.  A  3  per  cent,  solution 


DISINFECTANTS    AND    ANTISEPTICS.  2OI 

will  sometimes  kill  the  spores  of  anthrax  after  two  days' 
exposure  (see  Bacillus  anthracis,  Part  IV.)-  In  the  ab- 
sence of  spores  the  anthrax  bacillus  is  destroyed  by  a  I  per 
cent,  solution  in  one  hour.  The  less  resistant  pus  cocci  are 
destroyed  rapidly  by  a  2  per  cent,  solution.  Combination 
with  an  equal  proportion  of  hydrochloric  acid  enhances  the 
efficacy  of  carbolic  acid  to  a  marked  extent.  This  is  due 
to  the  prevention  of  albuminous  combinations,  thus  allow- 
ing- greater  penetration  of  the  disinfectant. 

Many  other  substances  closely  related  to  carbolic  acid 
are  used  and  possess  marked  germicidal  properties. 
Among  them  may  be  mentioned  creolin,  cresol  and  lysol. 
They  are  all  slightly  superior  to  carbolic  acid  in  actual 
germicidal  value. 

Aniline  Dyes. — Many  of  these  substances  possess  germ- 
icidal properties,  notably  pyoktanin  (methyl-violet).  A 
solution  of  1-5000  will  kill  the  anthrax  bacillus  in  two 
hours.  A  much  stronger  solution,  1-150,  is  required  to 
kill  the  typhoid  bacillus  in  the  same  time.  Malachite- 
green  is  said  to  possess  even  greater  germicidal  value  than 
pyoktanin.  Methylene-blue  also  possesses  considerable 
germicidal  power.' 

Formaldehyde. — A  gaseous  substance  placed  on  the 
market  in  a  40  per  cent,  aqueous  solution.  Remarkable 
claims  have  been  made  for  this  substance,  and  numerous 
investigations  have  shown  it  to  possess,  both  in  the  liquid 
and  gaseous  forms,  wonderful  disinfecting  power  under 
certain  conditions.  It  is  a  noticeable  fact  that  the  more 
recent  the  investigation  the  lower  the  value  placed  upon  it. 
In  solutions  of  i-iooo  an  exposure  of  twenty-four  hours  is 
necessary  to  destroy  the  staphylococcus  pyogenes  aureus, 
while  1-5000  is  sufficient  to  restrain  its  growth  (Slater 
and  Rideal).  Its  use  in  a  gaseous  form  as  a  house-disin- 


202  MANUAL    OF    BACTERIOLOGY. 

fectant  is  by   far   the  most   important   application   at  the 
present  time. 

Harrington's  investigations  have  shown  that  an  atmosphere  produced 
by  vaporizing  435  c.c.  of  formalin  (40  per  cent,  aqueous  solution  of 
the  gas)  in  1000  cubic  feet  of  air  space,  equivalent  to  i  quart  to  a  room 
15  feet  square  and  10  feet  high,  will  destroy  all  exposed  organisms  in 
half  an  hour ;  when  protected  by  one  fold  of  cotton-cloth,  an  exposure 
of  one  and  one-half  hours  is  necessary.  In  a  perfectly  dry  atmosphere 
the  gas  penetrates  slightly,  and  will  disinfect  through  one  layer  of 
cotton-cloth;  in  a  moist  atmosphere  no  penetration  can  be  obtained. 

In  vaporizing  the  gas  many  methods  have  been  em- 
ployed. Simple  evaporation  of  solutions  without  heat 
cannot  be  relied  upon,  for  the  solid,  polymerized  para- 
formaldehyde  is  easily  formed  under  these  circumstances. 
Better  results  can  be  obtained  with  the  aid  of  heat,  although 
polymerization  is  apt  to  occur  unless  evaporation  is  rapid. 
To  produce  the  best  results  it  has  been  found  necessary  to 
use  special  forms  of  lamps  or  generators  for  its  production, 
a  few  of  which  are  mentioned  below. 

Sanitary  Construction  Company's  Lamp. — This  lamp 
consists  of  a  tank  to  hold  the  formaldehyde  solution,  and  a 
spiral  tube  by  which  the  solution  is  slowly  conducted  through 
a  flame  and  vaporized.  The  necessary  amount  of  solution 
is  placed  in  the  tank  and  the  apparatus  started,  outside 
the  room,  the  gas  being  conducted  through  the  keyhole  by 
a  suitable  tube. 

Trillat  Autoclave. — A  small  silver-lined  pressure-boiler, 
fitted  with  lamp,  safety-valve,  pressure-gauge,  thermome- 
ter and  escapement-tube.  The  necessary  amount  of  form- 
aldehyde solution  is  placed  within  the  apparatus,  together 
with  an  equal  amount  of  20  per  cent,  solution  of  calcium 
chloride ;  the  addition  of  the  latter  salt  is  to  prevent  forma- 
tion of  the  solid  polymeric  modification,  the  so-called  para- 
form.  The  autoclave  is  closed  and  heated  from  below  to 
a  temperature  of  135°  C.  The  escapement-valve  is  then 
opened  carefully  and  the  gas  allowed  to  enter  the  room 


DISINFECTANTS    AND    ANTISEPTICS.  203 

slowly  through  the  escapement-tube,  which  has  meanwhile 
been  passed  through  the  keyhole.  About  thirty  minutes 
are  required  to  discharge  all  the  gas  from  500  c.c.  of  solu- 
tion. If  the  temperature  has  not  been  allowed  to  go  above 
135°  C.  the  gas  will  contain  but  little  moisture  and  possess 
its  maximum  efficiency. 

Schering  Lamp. — This  lamp  is  intended  to  utilize  para- 
form  or  para-formaldehyde,  a  polymeric  modification  of 
formaldehye,  occurring  as  a  white  salt.  It  is  decompos- 
able by  heat,  yielding  formaldehyde  gas.  It  is  placed  on 
the  market  in  the  form  of  tablets,  each  one  of  which  yields 
a  definite  amount  of  gas.  The  lamp  consists  of  a  small 
iron  tray  for  the  reception  of  tablets,  and  so  arranged  above 
the  heating-apparatus  that  sufficient  draught  is  created  to 
carry  off  the  gas  as  rapidly  as  formed.  In  operating,  a 
sufficient  number  of  tablets  are  placed  on  the  tray,  the  lamp 
lighted  and  placed  in  the  room  to  be  disinfected. 

Methyl-Alcohol  Lamps. — Several  of  these  lamps  are  on 
the  market,  all  operating  on  the  well-known  principle  of 
the  oxidation  of  wood-alcohol  to  formaldehyde  when  the 
alcohol  is  vaporized  by  projection  against  a  heated,  plati- 
nized, asbestos  disk.  In  operating  such  an  apparatus,  the 
alcohol  is  lighted  until  the  asbestos  disk  becomes  hot.  The 
flame  is  then  extinguished;  the  heat  from  the  disk  is  suffi- 
cient to  vaporize  the  alcohol,  which  undergoes  oxidation 
and  keeps  the  disk  at  a  red  heat.  When  the  apparatus  is 
operating  in  a  satisfactory  manner  the  room  is  closed  and 
disinfection  allowed  to  proceed.  It  must  be  said,  however, 
that  it  is  difficult  to  estimate  or  control  the  amount  of  form- 
aldehyde evolved  in  generators  of  this  type. 

Formaldehyde  Candles. — Mixtures  of  para-formaldehyde 

and  paraffin  or  other  combustibles,  which  may  be  moulded 

into  candles,  each  enclosed  in  a  tin  case,  make  a  convenient 

apparatus  to  generate  formaldehyde  gas  for  room  disinfec- 

18 


204  MANUAL    OF    BACTERIOLOGY. 

tion.  The  candle  is  placed  in  a  suitable  fire-proof  dish, 
it  is  then  ignited,  and  generation  of  the  gas  is  allowed  to 
proceed  in  the  tightly  closed  room. 

Sulphur  Dioxide. — This  substance  is  used  extensively 
for  house  disinfection,  and  is  usually  prepared  by  burning 
sulphur.  Much  difference  of  opinion  exists  regarding  the 
value  of  it  as  a  disinfectant.  The  spores  of  anthrax  are 
not  killed  by  several  days'  exposure  to  the  liquefied  gas. 
Anthrax  and  other  bacilli  are  destroyed  in  thirty  minutes 
when  exposed  on  moist  threads  in  an  atmosphere  contain- 
ing one  volume  per  centum  of  the  gas.  An  exposure  of 
twenty-four  hours  in  an  atmosphere  containing  four  vol- 
umes per  centum  of  the  gas  will  destroy  the  organisms  of 
typhoid  fever,  diphtheria,  cholera  and  tuberculosis.  The 
presence  of  moisture  greatly  enhances  the  activity  of  the 
disinfectant,  owing  to  the  formation  of  the  more  energetic 
sulphurous  acid. 

For  the  destruction  of  insects,  such  as  mosquitoes,  this 
agent  is  superior  to  formaldehyde.  Its  application  for  this 
purpose  is  important  in  preventing  the  spread  of  yellow 
fever  and  malaria. 

In  practice,  at  least  3  pounds  of  sulphur  per  1000  cubic 
feet  should  be  used,  and  moisture  must  be  present.  This 
latter  requirement  can  be  fulfilled  by  evaporating  several 
quarts  of  water  within  the  tightly  closed  room  just  prior  to 
generating  the  gas.  In  using  powdered  or  flowers  of  sul- 
phur, the  necessary  amount  is  placed  on  a  bed  of  sand  or 
ashes  in  an  iron  pot,  which  should  rest  on  a  couple  of 
bricks  in  a  pan  or  other  vessel  containing  an  inch  or  two 
of  water.  The  sulphur  is  ignited  by  means  of  some  glow- 
ing coals,  or  by  moistening  with  alcohol  and  applying  a 
a  match.  Difficulty  is  often  experienced  in  keeping  the  sul- 
phur burning,  and  for  this  reason  it  is  surer  and  more  con- 
venient to  use  the  so-called  sulphur  candles  now  on  the 


DISINFECTANTS    AND    ANTISEPTICS.  2C«5 

market.  In  operating  with  these,  a  sufficient  number  are 
placed  on  bricks  in  a  pan  of  water  and  the  wicks  lighted. 
Liquefied  sulphur  dioxide  may  be  used,  and  can  now  be 
obtained  in  convenient  tin  receptacles  containing  a  suffi- 
cient quantity  for  the  disinfection  of  an  ordinary  room. 
The  can  is  opened  by  cutting  through  a  soft  metal  tube 
projecting  from  the  top.  The  fluid  vaporizes  at  the  room 
temperature,  and  it  is  simply  necessary  to  place  the  can  in  a 
convenient  porcelain  dish  and  allow  the  fluid  to  evaporate. 

Sulphur  dioxide  is  objectionable  on  account  of  its  lack 
of  power  when  dry,  and  on  account  of  its  corrosive  action 
on  metal  and  its  bleaching  effect  on  hangings  and  draperies 
in  the  presence  of  moisture;  it  is,  therefore,  preferable  to 
use  formaldehyde  when  possible. 

Chlorine. — A  very  active  gaseous  disinfectant,  particu- 
larly in  the  presence  of  moisture.  An  atmosphere  con- 
taining i  per  cent,  of  the  dry  gas  is  fatal  to  anthrax  spores 
in  three  hours.  The  anthrax  bacillus  is  killed  in  twenty- 
four  hours  by  exposure  to  a  moist  atmosphere  containing 
the  gas  in  the  proportion  of  1-2500.  The  bacillus  of  tuber- 
culosis is  killed  by  an  exposure  of  one  hour  to  a  moist  atmos- 
phere containing  the  gas  in  the  proportion  of  1-200.  Ex- 
tremely minute  quantities  in  solution  will  prevent  the 
development  of  putrefactive  organisms.  The  substance 
has  been  used  for  house  and  ship  disinfection,  but  is  now 
seldom  employed  on  account  of  its  extremely  irritating 
properties  and  the  difficulty  of  handling  it. 

Bromine. — Used  in  the  gaseous  and  liquid  form.  The 
dry  vapor  possesses  but  little  disinfectant  power;  when 
moist  it  is  much  more  efficient.  In  saturated  aqueous  so- 
lution it  will  kill  the  anthrax  bacillus  in  twenty-four  hours. 

Calcium  Hypochlorite,  usually  known  as  Chloride  of 
Lime. — This  is  a  most  practical  and  valuable  disinfectant, 
depending  for  its  efficiency  on  the  available  chlorine  con- 


206  MANUAL    OF    BACTERIOLOGY. 

tained  in  it.  Its  alkalinity  favors  penetration,  and  for 
many  purposes  it  cannot  be  excelled.  A  I  per  cent,  solu- 
tion will  destroy  anthrax  spores  in  one  hour.  A  solution 
of  the  same  strength  will  disinfect  typhoid  stools  in  ten 
minutes. 

Lime. — The  addition  of  o.i  per  cent,  of  unslaked  lime 
to  fluid-cultures  of  the  typhoid  bacillus  and  cholera  spiril- 
lum will  render  them  sterile  in  four  or  five  hours.  Ty- 
phoid dejecta  are  sterilized  in  six  hours  by  the  addition  of 
3  per  cent,  of  slaked  lime;  the  addition  of  6  per  cent,  will 
accomplish  the  same  result  in  two  hours.  A  convenient 
form  for  practical  use  is  an  aqueous  mixture  containing  20 
per  cent,  of  lime — so-called  milk  of  lime.  Typhoid  and 
cholera  dejecta  are  sterilized  in  one  hour  after  the  addition 
of  20  per  cent,  of  this  mixture.  In  practice  it  is  safer  to 
use  a  considerable  excess  of  lime.  From  the  foregoing  facts 
it  would  seem  probable  that  lime  or  whitewash  as  ordi- 
narily applied  would  possess  disinfectant  properties.  Ex- 
perimental work  has  demonstrated  this  to  be  a  fact.  The 
organisms  of  anthrax,  glanders  and  the  pus  cocci  were 
destroyed  within  twenty-four  hours  by  one  application. 
For  spore-forming  organisms  and  the  bacillus  of  tubercu- 
losis the  power  is  not  so  great,  the  latter  organism  not 
being  destroyed  by  three  applications  of  the  whitewash. 
This  is  due,  perhaps,  to  the  large  amount  of  fatty  matter 
in  the  bacillus  of  tuberculosis,  and  suggests  the  possibility 
of  enhancing  the  efficacy  of  the  lime  by  the  addition  of  a 
small  proportion  of  caustic  alkali. 

Hydrogen  Peroxide. — This  substance  is  placed  on  the 
market  in  solutions  varying  in  strength  from  10  to  30  vol- 
umes;  the  mode  of  expression  indicating  that  correspond- 
ing solutions  will  liberate  ten  to  thirty  times  their  volume 
of  oxygen  when  appropriately  treated.  It  possesses  the 
property  of  rapidly  oxidizing  purulent  secretions,  and  on 


DISINFECTANTS    AND    ANTISEPTICS.  2OJ 

this  account  is  much  used  for  cleansing  infected  wounds. 
It  deteriorates  in  strength  so  rapidly  that  only  fresh  solu- 
tions of  known  strength  should  be  used. 

Potassium  Permanganate. — Koch  asserts  that  a  3  per 
cent,  solution  will  destroy  anthrax  spores  in  twenty-four 
hours,  but  that  a  i  per  cent,  solution  cannot  be  depended 
upon  to  kill  pathogenic  organisms.  Its  disinfectant  value  in 
practice  is  very  low  on  account  of  its  ready  decomposition 
by  inert  material.  In  the  dilute  solutions  usually  used  for 
medicinal  injections  and  irrigations  no  disinfectant  action 
occurs. 

lodoform. — This  substance  possesses  little  if  any  disin- 
fectant power.  It  is  mildly  antiseptic  in  moist  wounds, 
due  to  the  gradual  liberation  of  small  quantities  of  iodine. 

Boric  Acid. — This  material  possesses  practically  no  dis- 
infectant power.  It  is  a  mild  antiseptic  when  applied  as 
an  undiluted  powder  to  wounds.  A  saturated  aqueous 
solution  is  much  used,  and  is  weakly  antiseptic. 

Essential  Oils. — Many  of  these  bodies  possess  germi- 
cidal  value,  notably  the  oils  of  cinnamon  and  cloves.  The 
oil  of  mustard  is  also  a  valuable  disinfectant,  but  so  irri- 
tating that  the  pure  oil  cannot  be  used.  The  use  of  pow- 
dered mustard  in  the  autopsy-room  will  remove  the  foul 
odor  from  the  hands  more  rapidly  and  completely  than  any 
other  means. 

Coal  Oil  or  Petroleum. — While  the  disinfectant  value  of 
this  substance  is  slight,  its  use  in  destroying  the  larvae  of 
insects,  such  as  the  mosquito,  has  given  it  an  important 
position  in  preventing  the  spread  of  malaria  and  yellow 
fever.  A  small  amount  poured  on  a  stagnant  pool  rapidly 
spreads  over  the  surface,  and  effectually  destroys  such 
larvae. 

Ferrous  Sulphate  (Copperas). — This  salt  has  been  much 
used,  but  possesses  only  feeble  disinfectant  powers.  A  3 


208  MANUAL    OF    BACTERIOLOGY. 

per  cent,  solution  requires  three  days  to  kill  the  bacillus  of 
typhoid  fever.  On  account  of  its  affinity  for  ammonia  and 
sulphides  it  is  an  efficient  deodorizer  for  temporary  use,  but 
cannot  be  relied  upon  to  kill  the  bacteria  producing  the 
noxious  gases. 

Cupric  Sulphate  (Blue  Vitriol}. — This  salt  is  quite  an 
efficient  disinfectant.  In  a  solution  of  1-3000  the  spirillum 
of  cholera  is  destroyed  in  ten  minutes.  A  5  per  cent,  solu- 
tion will  kill  the  typhoid  bacillus  in  ten  minutes.  A  solu- 
tion of  from  2  to  3  per  cent,  in  strength  can  be  relied  upon 
to  destroy  all  pathogenic  organisms  that  do  not  form  spores. 

Zinc  Sulphate. — This  salt  is  a  very  feeble  disinfectant. 
Pus  cocci  are  not  destroyed  in  two  hours  by  a  20  per  cent, 
solution.  As  a  deodorizer  it  has  about  the  same  value  and 
acts  in  the  same  way  as  ferrous  sulphate. 

Zinc  Chloride. — A  2  per  cent,  solution  will  kill  pus  cocci 
after  an  exposure  of  two  hours.  It  is  therefore  a  much 
more  powerful  disinfectant  than  the  sulphate. 

Disinfection  of  Dejecta  and  Urine. — A  4  per  cent,  solu- 
tion of  calcic  hypochlorite  (chloride  of  lime)  is  most  effi- 
cient and  rapid  for  this  purpose.  A  convenient  solution 
contains  6  ounces  of  the  salt  to  i  gallon  of  water.  The 
excreta  should  be  received  in  a  suitable  vessel  and  imme- 
diately mixed  with  an  equal  bulk  of  the  disinfectant.  The 
contents  of  the  vessel  should  be  allowed  to  stand  for  one 
hour  before  emptying.  A  20  per  cent,  milk  of  lime  is  just 
as  efficient,  and  possesses  the  advantage  of  cleanliness  and 
lack  of  odor.  It  should  be  used  in  the  same  quantity  and 
allowed  to  act  for  the  same  length  of  time.  A  5  per  cent, 
solution  of  carbolic  acid  may  be  used,  but  should  be  allowed 
to  act  for  at  least  four  hours. 

Disinfection  of  Sputum. — The  chemical  disinfection  of 
tuberculous  sputum  is  somewhat  difficult  on  account  of  the 
large  amount  of  albumin  in  it  and  the  fatty  matter  associated 


DISINFECTANTS    AND    ANTISEPTICS.  2(X) 

with  the  bacillus  of  tuberculosis.  Dilute  solutions  of  bi- 
chloride of  mercury  are  apt  to  be  decomposed  and  rendered 
inert  by  the  albumin.  Carbolic  acid  is  open  to  the  same 
objection,  but  its  combination  with  hydrochloric  acid  can 
be  used  successfully  in  a  strength  of  5  per  cent.  each. 
Milk  of  lime  cannot  be  relied  upon  for  this  purpose.  A 
4  per  cent,  solution  of  calcic  hypochlorite  (chloride  of  lime) 
is  the  best  for  general  use,  and  the  spit-cup  should  be  kept 
nearly  full  of  this  solution.  Sputum  may  also  be  disinfected 
by  exposure  to  the  action  of  steam  in  the  steam  sterilizer 
or  by  boiling  for  15  minutes.  If  napkins  or  old  pieces  of 
cloth  are  used  for  the  reception  of  sputum  they  may  be 
immediately  destroyed  in  a  fire. 

Disinfection  after  Postmortems. — After  autopsies  on 
infectious  cases  it  is  necessary  to  disinfect  the  table  and 
fluid  products  coming  from  it  prior  to  emptying  into  the 
sewer.  The  table  may  be  successfully  disinfected  by  a 
liberal  sprinkling  with  4  per  cent,  calcic  hypochlorite  solu- 
tion. All  fluids  should  be  treated  with  an  equal  quantity 
of  the  same  solution.  The  table  should  not  be  cleaned  for 
at  least  one  hour  after  application  of  the  disinfectant.  The 
same  rule  applies  to  the  disinfection  of  the  fluids — an  ex- 
posure of  at  least  one  hour  to  the  disinfectant  before  final 
disposition. 

The  Cadaver  in  Contagions  Diseases. — In  cases  of  death 
from  a  contagious  disease  all  the  orifices  of  the  body  should 
be  packed  with  cotton  soaked  in  a  strong  solution  ( i  to  500) 
of  bichloride  of  mercury,  the  skin  washed  with  a  i  to  1000 
solution,  and  the  cadaver  wrapped  in  a  sheet  wet  with  the 
same.  The  funeral  should  be  private  and  the  body  dis- 
posed of  within  twenty-four  hours,  preferably  by  cremation. 

House  Disinfection. — After  infectious  disease  it  is  essen- 
tial that  the  house  or  the  apartment  in  which  the  patient 
has  been  confined  should  be  disinfected.  It  is  rarely  neces- 


2IO  MANUAL    OF    BACTERIOLOGY. 

sary  to  carry  out  the  process  in  more  than  two  rooms ;  but 
should  it  be  so,  the  process  can  be  applied  to  the  whole 
house. 

After  thorough  bathing  of  the  patient,  preferably  with 
an  antiseptic  soap,  the  individual  should  be  wrapped  in  a 
clean  sheet  and  removed  to  a  clean  room.  All  articles  or 
materials  that  are  of  little  value  should  be  destroyed.  All 
bedding,  towels  and  the  like  should  be  placed  in  wooden 
tubs  and  covered  with  a  i-iooo  solution  of  bichloride  of 
mercury.  The  room  should  then  be  made  as  nearly  air- 
tight as  possible ;  this  can  be  accomplished  by  pasting  strips 
of  paper  over  registers,  cracks,  spaces  between  window- 
sashes  and  the  like.  Formaldehyde  gas  is  then  passed 
through  the  keyhole  into  the  room  (or  it  may  be  generated 
by  formaldehyde  candles)  in  sufficient  quantity  to  destroy 
the  infectious  element.  The  room  should  be  sealed  for  at 
least  twelve  hours,  after  which  time  it  may  be  opened  and 
aired.  The  process  is  completed  by  washing  all  exposed 
surfaces  in  the  room  with  i-iooo  bichloride  of  mercury. 
This  latter  requirement  is  not  essential  if  the  gaseous  disin- 
fection has  been  complete,  but  since  we  have  no  absolute 
knowledge  on  this  point,  the  secondary  washing  should  be 
carried  out.  This  method  can  be  considered  reliable  for 
surface  disinfection,  but  for  the  interior  of  mattresses  and 
stuffed  furniture-cushions  it  is  not  certain.  In  all  cases 
where  absolute  disinfection  is  demanded,  such  articles  must 
be  ripped  apart  and  loosely  exposed  to  the  gas.  They  may 
be  disposed  of  by  fire  or  sterilized  by  steam  under  pressure. 
The  latter  method  must  necessarily  be  a  matter  of  municipal 
control,  and  can  only  be  carried  out  by  means  of  suitable 
apparatus  in  the  hands  of  a  municipal  disinfecting  corps. 
Instead  of  formaldehyde,  sulphur  dioxide  may  be  used  for 
room  disinfection,  but  in  the  light  of  present  knowledge  the 
formaldehyde  method  is  superior. 


PREPARATION    OF    INSTRUMENTS,    ETC.  211 


CHAPTER  IX. 

THE     PREPARATION     OF     INSTRUMENTS,     LIGATURES,     DRESS- 
INGS,   ETC.,    FOR    SURGICAL    PURPOSES.1 

THE  purpose  of  this  chapter  is  to  explain  the  application 
of  the  principles  set  forth  on  the  preceding  pages  to  sur- 
gical technique.  It  has  been  shown  that  all  objects  about 
us  may  have  bacteria  on  them,  and  that  bacteria  are  pres- 
ent on  all  the  surfaces  of  our  bodies  that  come  in  contact 
with  the  air.  All  the  care  that  is  needed  in  working  with 
bacteria  in  the  laboratory,  and  more,  must  be  exercised  in 
surgical  operations.  Everything  that  has  not  been  steril- 
ized must  be  regarded  as  having  the  possibilities  of  infec- 
tion in  it.  After  the  hands  have  been  cleansed,  if  they  touch 
the  clothing  or  furniture,  they  must  be  cleansed  again. 
If  a  sterilized  instrument  falls  on  the  floor,  it  must  be  ster- 
ilized again.  The  same  applies  to  dressings,  sponges,  liga- 
tures, or  anything  which  is  to  be  used  about  the  wound. 

The  value  of  chemical  germicides  has  probably  been  over- 
rated in  the  past.  They  are  used  only  to  destroy  the  bac- 
teria on  living  tissues  and  on  articles  that  would  be  damaged 
by  heat.  They  give  less  reliable  results  than  boiling. 
Wherever  boiling  or  steam  sterilization  is  permissible,  it 
should  be  used.  With  materials  that  may  contain  a  small 
quantity  of  substance  in  which  bacteria  can  grow,  the  frac- 
tional method  of  sterilization  should  be  used  (see  page  63). 
With  glass  and  metallic  objects,  obviously  a  single  boiling 
can  accomplish  as  much  as  boiling  on  three  consecutive  days. 

1  By  Marshall  Clinton,  M.D.,  Instructor  in  Clinical  Surgery,  Medical 
Department,  University  of  Buffalo. 


212  MANUAL    OF    BACTERIOLOGY. 

The  failures  in  the  practice  of  aseptic  surgery  are  gen- 
erally due  to  the  hands  of  the  operator  and  assistants  and 
the  skin  of  the  patient. 

The  following  formulae  have  been  selected  from  the  many 
published  as  they  are  successfully  used  by  many  surgeons, 
and  meet  the  theoretical  grounds  of  bacteriology  as  far  as 
is  possible  with  our  present  knowledge. 

Sterilization  of  Hands. — There  is  no  known  method  for 
perfect  sterilization  of  the  human  skin.  A  close  approach 
to  sterility  is  reached  by  any  one  of  the  methods  that  has 
as  its  basis  mechanical  cleanliness. 

Park's  method  :  ( i )  Hands  and  forearms  are  thoroughly 
rubbed  with  a  mixture  of  green  soap  and  cornmeal,  which 
serves  to  remove  all  the  loose  dirt  and  epithelium.  Rinse 
carefully  until  hands  and  forearms  are  clean.  (2)  A  paste 
of  mustard  flour  and  cold  water  is  rubbed  into  the  hands 
and  forearms  until  they  begin  to  sting.  (3)  Rinse  in  run- 
ning sterile  water;  then  soak  in  a  hot  i-iooo  bichloride  of 
mercury  solution  for  a  few  minutes,  the  fluid  being  well 
rubbed  into  the  skin. 

FUrbringer's  method :  ( i )  Thorough  scrubbing  of  the 
hand  and  forearms  with  soft  soap,  water  and  a  nail-brush 
for  at  least  three  minutes,  especial  attention  being  paid  to  the 
nails.  (2)  Removal  of  all  fat  and  debris  by  rubbing  hands 
and  forearms  while  immersed  in  95  per  cent,  alcohol.  (3) 
Rinsing  of  hands  and  forearms  in  a  i-iooo  bichloride  of 
mercury  solution,  rubbing  the  fluid  well  into  the  skin. 

Schatz's  method  :  ( i )  Hands  and  forearms  are  cleansed 
by  brisk  scrubbing  with  soft  soap  and  a  clean  brush  for 
from  three  to  five  minutes.  (2)  Soaking  in  saturated  solution 
of  permanganate  of  potassium  at  a  temperature  of  110°  F. 
until  the  hands  and  forearms  are  a  deep  mahogany  brown. 
(3)  Immersion  in  a  saturated  solution  of  oxalic  acid,  tem- 
perature of  110°  F.  until  the  skin  is  entirely  decolorized. 


PREPARATION    OF    INSTRUMENTS,,    ETC.  213 

(4)  Rinsing  with  sterile  lime  water  to  rid  of  excess  of  acid. 

(5)  Washing  in  i-iooo  bichloride  of  mercury  solution  for 
one  minute. 

Weir's  method :  ( i )  Hands  and  forearms  are  scrubbed 
as  in  other  methods.  (2)  A  scant  tablespoonful  of  chlori- 
nated lime  is  moistened  with  enough  warm  water  to  make  a 
thick  paste.  This  is  carefully  rubbed  into  hands  and  fore- 
arms. (3)  A  piece  of  carbonate  of  soda  one  inch  square  and 
one-half  inch  thick  is  crushed  and  rubbed  into  the  paste. 
From  three  to  five  minutes  are  thus  employed.  (4)  Rinsing 
in  sterile  water  and  washing  in  a  solution  of  £  of  I  per  cent, 
of  ammonia,  removes  the  odor  of  chlorine. 

E.  R.  McGuire1  states  that  prolonged  scrubbing  with  frequent  changes 
of  brushes  in  running  sterile  water  will  give  the  nearest  approach 
to  sterility.  The  use  of  antiseptics  on  the  hands  is  not  to  be  relied 
upon,  for  their  precipitation  by  chemicals  or  normal  tissue  fluids  may 
break  up  their  combination  with  bacteria  that  were  considered  inactive 
or  dead,  but  are  not  so  in  reality.  He  suggests  the  use  of  hot-air,  by 
cabinet-bath  or  Kelly  hot-air  apparatus,  to  "  sweat "  out  of  the  glands 
in  the  skin  as  much  as  possible  of  their  contents  before  the  skin  is 
cleansed. 

Prolonged  soaking  of  the  skin  in  a  soap  poultice  or  strong 
antiseptic  may  damage  and  irritate  the  tissues,  so  that  it  is 
not  advisable  to  prepare  the  field  of  operation  more  than 
twelve  or  twenty-four  hours  before  the  time  set  for  an 
operation. 

Maylard2  recommends  the  sterilization  of  the  skin  by 
inunctions  of  oleate  of  mercury.  The  method  employed  is 
as  follows  :  ( i )  Cleanse  the  skin  in  the  usual  way  with  soap 
and  water.  (2)  Anoint  freely  and  widely  with  hydrated 
lanolin-oleate  of  mercury,  20  per  cent.,  and  rub  in;  smear 
a  piece  of  gauze  with  the  same  and  leave  until  a  second 
inunction  is  performed  twelve  hours  later.  Every  case 

1  American  Medicine,  February  28,  1903. 

2  Annals  of  Surgery,  January,  1902. 


214  MANUAL    OF    BACTERIOLOGY. 

should  be  treated  for  at  least  twenty-four  hours  before  opera- 
tion; preferably  forty-eight  hours  should  be  given,  with  at 
least  two  separate  periods  of  "  rubbing  in  "  for  about  ten 
minutes  on  each  occasion.  (3)  On  the  operating  table  the 
piece  of  gauze  is  removed,  and  the  superfluous  ointment 
rubbed  off  with  a  piece  of  sterile  gauze. 

To  Prepare  the  Field  of  Operation. — Wash  with  green 
soap  and  water,  scrubbing  thoroughly  and  carefully,  paying 
particular  attention  not  to  scrub  hard  enough  to  render  the 
skin  tender  or  to  make  abrasions.  Shave  parts  with  clean 
razor.  Wash  with  ether  and  alcohol,  to  remove  debris  and 
epithelium,  and  cover  with  a  sterile  towel.  If  the  skin  of  the 
patient  is  thick  a  soap  poultice  may  be  left  on,  care  being 
taken  to  see  that  the  skin  does  not  become  macerated.  After 
the  patient  is  anesthetized  the  field  is  briskly  scrubbed  with 
sterile  brushes,  soap,  and  water,  washed  with  i-iooo 
bichloride  of  mercury  solution  and  covered  with  sterile 
towels. 

It  is  important  to  remember  that  during  an  operation 
patient,  operator  and  assistants,  may  perspire  and  that  in  this 
way  fresh  masses  of  bacteria  from  the  deeper  parts  of  the 
glands  may  be  brought  to  the  surface  of  the  skin.  Careful 
attention  must  be  paid  to  maintaining  cleanliness  during  an 
operation.  The  patient's  skin  is  kept  covered  with  sterile 
towels,  changed  as  often  as  they  become  soiled.  For  the 
surgeon's  and  assistant's  hands  rubber  gloves  do  this  per- 
fectly. If  an  operator  or  assistant  finds  that  the  hands  per- 
spire during  an  operation  the  use  of  rubber  gloves  becomes 
essential.  Rubber  gloves  may  be  sterilized  by  boiling. 

Instruments  are  best  sterilized  by  contact  with  super- 
heated steam,  or  steam  under  pressure  for  ten  minutes,  or 
by  boiling  in  a  I  per  cent,  carbonate  of  soda  solution.  If 
soda  is  unavailable  use  water  that  is  actively  boiling,  as  this 
avoids  spotting  and  rusting  of  the  instruments.  Imme- 


PREPARATION    OF    INSTRUMENTS,    ETC.  215 

diately  after  use  instruments  should  be  thoroughly  scrubbed 
with  a  brush  and  washed  with  soap  and  hot  water  and 
boiled,  before  being  replaced  in  the  instrument  case. 

The  practice  of  passing  an  instrument  through  a  flame 
a  few  times  cannot  be  relied  on  to  destroy  the  bacteria  that 
may  be  present. 

Aspirating  syringes,  needles,  trocars,  drainage  tubes  and 
glass  nozzles  are  best  sterilized  by  boiling  for  ten  minutes. 
If  syringes  have  leather  washers  (which  should  be  avoided) 
they  may  be  cleansed  with  hot  water  and  soap,  rinsed  with 
alcohol,  filled  and  refilled  with  boiling  water  ten  or  more 
successive  times,  and  placed  in  a  1-40  carbolic  acid  solution. 

Instrument  trays,  ligature  dishes,  basins  for  sponges,  etc., 
are  to  be  sterilized  by  boiling  for  ten  minutes,  and  protected 
from  dust  with  sterile  towels. 

Catgut  is  made  from  the  intestines  of  sheep,  and  sheep  are 
subject  to  anthrax  infection,  while  tetanus  bacilli  may  occur 
in  the  intestine.  Therefore,  catgut  must  be  sterilized  by 
some  method  that  will  kill  the  spores  of  these  organisms  if 
present  (see  Bacilli  of  anthrax  and  tetanus,  Part  IV.). 
There  are  many  methods  devised  for  the  preparation  of 
sterile  catgut  that  have  as  a  basis  an  incorporation  within 
the  catgut  of  some  antiseptic.  They  are  open  to  the  objec- 
tion that  any  antiseptic  introduced  into  the  tissues  acts  as  an 
irritant,  aside  from  the  fact  that  organisms  may  be  liberated 
from  partially  absorbed  catgut.  This  is  seen  in  cases  of 
late  suppuration — ten  to  fifteen  days  after  an  operation. 

Catgut  comes  in  sizes  from  double  zero  up  to  No.  8.  The 
sizes  mostly  used  are  o  to  4.  Catgut  when  ready  for  use 
should  be  smooth,  soft,  pliable,  and  very  strong ;  wiry  catgut 
is  apt  to  cut  through  tissues. 

Cumol  method.1  The  catgut  is  rolled  on  glass  spools,  and 
these  put  in  a  glass  beaker.  The  bottom  of  the  beaker  is 

1  Clark  and  Miller,  Bulletin  Johns  Hopkins  Hospital,  Vol.  XL,  Sep- 
tember, 1900. 


2l6  MANUAL    OF    BACTERIOLOGY. 

covered  with  a  layer  of  cotton  on  which  the  catgut  rests. 
The  beaker  is  heated  with  a  Bunsen  burner  over  a  sand- 
bath.  The  top  of  the  beaker  is  covered  with  a  piece  of 
cardboard.  Through  a  hole  in  the  center  of  the  cardboard 
a  thermometer  passes.  Heat  is  now  applied  to  the  sand- 
bath,  and  the  temperature  of  the  catgut  slowly  raised  to 
80°  C.  In  this  manner  all  moisture  is  driven  out  of  the 
catgut.  This  degree  of  heat  is  maintained  for  one  hour. 
Cumol1  at  a  temperature  of  100°  C.  is  now  added  to  the 
beaker,  completely  covering  the  catgut.  The  beaker  should 
be  covered  with  copper-wire  netting  to  prevent  ignition  of 
the  cumol,  which  is  very  inflammable.  The  temperature  is 
then  increased  to  165°  C.,  and  kept  at  this  point  for  one 
hour.  The  fluid  is  now  poured  off,  and  the  catgut  allowed 
to  dry  in  the  beaker  on  the  sand-bath  at  a  temperature  of 
1 00°  C.  for  two  hours.  It  is  then  transferred  to  sterile  jars 
or  test-tubes  until  needed,  or  it  may  be  preserved  in  sterile 
alcohol. 

Formaldehyde  catgut.2  Three-quarter-inch  glass  spools 
are  notched  on  each  flange.  The  catgut  is  wound  upon  the 
spool  tightly  in  one  layer,  and  evenly,  the  ends  passing  over 
the  flange  of  the  spool  in  the  notch;  the  longer  end,  after 
passing  through  the  notch,  goes  through  the  barrel  of  the 
spool  and  is  securely  tied  to  the  shorter  end  which  has 
passed  over  the  other  notched  flange.  By  thus  winding  the 
gut  there  will  be  enough  for  one  or  two  ligatures  or  sutures 
of  good  length.  Gut  prepared  by  this  process  tends  to 
contract  forcibly,  and  on  account  of  this  strain  must  be 
held  securely  or  it  will  shrink  and  be  useless.  The  object 
of  winding  in  a  single  layer,  evenly,  is  to  prevent  over- 
lapping or  crossing  of  one  strand  over  another.  If  in 

1  Cumol  is  a  fluid  hydrocarbon,  with  a  boiling  point  somewhat  above 
165°  C.  It  dissolves  the  fat  in  catgut.  After  boiling  it  has  a  brown 
color. 

2Frederick,  American  Journal  Obstetrics,  Vol.  89,  1899. 


PREPARATION    OF    INSTRUMENTS,    ETC. 

the  process  of  soaking  in  formaldehyde  and  the  consequent 
shrinking,  one  strand  crosses  another,  the  one  next  to 
the  glass  will  be  so  pressed  upon  as  to  prevent  hardening 
at  that  point.  When  the  gut  is  boiled  later  in  water, 
that  point  will  gelatinize  and  break  at  the  least  strain. 
Formaldehyde  comes  in  a  40  per  cent,  solution.  We  use  a 
3  per  cent,  solution,  pouring  one  part  of  the  40  per  cent, 
formaldehyde  solution  and  thirteen  parts  of  water  into  a 
wide-mouthed  bottle.  Immerse  the  spools  in  this  solution 
for  periods  of  time  varying  with  the  size  of  the  gut.  No.  o 
is  left  in  one  hour.  Nos.  i,  2  and  3  are  given  three,  five, 
and  seven  hours  respectively.  If  left  too  long  in  the  solu- 
tion the  gut  will  become  too  hard,  too  brittle,  and  the 
strength  will  be  impaired.  Wash  in  running  water  for  a 
longer  time  than  it  was  in  the  formaldehyde  solution.  Up 
to  this  time  the  gut  has  not  been  sterilized.  It  has  under- 
gone a  chemical  change  whereby  it  may  be  boiled  without 
spoiling  it.  The  sterilization  of  the  gut  consists  in  boiling 
for  fifteen  minutes,  with  the  receptacles  in  which  it  is  to  be 
kept.  With  sterile  forceps  place  the  spools,  each  size  by 
itself,  in  wide-mouthed-ground-glass-stoppered  bottles  or  in 
rubber-sealing  fruit  jars,  sterilized  by  boiling.  Pour  over 
the  gut  clean  95  per  cent,  alcohol  with  8  to  10  per  cent,  of 
glycerine.  To  sterilize  the  glycerine  it  should  be  placed  in 
a  bottle  in  water  and  raised  to  the  temperature  of  boiling 
water  for  half  an  hour. 

To  make  chromicizcd  catgut  wind  the  spools  as  before. 
Place  the  spools  in  a  solution  of :  bichromate  of  potassium, 
1.5  grammes;  glycerine  and  carbolic  acid  each  10  c.c. ;  water 
I  liter.  Allow  them  to  remain  in  this  solution  for  twenty- 
four  hours.  Take  out  and  drain,  allowing  them  to  dry  for  a 
few  hours.  Then  place  in  the  formaldehyde  solution  and 
put  through  the  same  process  as  with  formaldehyde  catgut. 

Kangaroo  tendon,  owing  to  its  slow  absorption,  is  used  as 


2l8  MANUAL    OF    BACTERIOLOGY. 

a  heavy  retaining  suture,  and  is  prepared  by  washing  the 
strands  in  ether  to  free  from  fat.  Soak  in  a  4  per  cent,  solu- 
tion of  chromic  acid  for  twenty-four  hours.  Then  sterilize 
by  the  cumol  method. 

Silk  may  be  sterilized  by  the  fractional  method  (see  p.  63) 
as  this  does  not  impair  the  strength  as  does  boiling. 

Silkworm  gut  is  prepared  by  steam  sterilization  by  the 
fractional  method  or  by  boiling  in  plain  water  for  one  half 
hour.  It  should  not  be  boiled  in  soda  solution  as  this  spoils 
the  gut. 

Horsehair  strands  are  cut  into  two-foot  lengths,  washed 
with  soap  and  water  and  sterilized  with  steam  by  the  frac- 
tional method.  They  make  a  very  fine  suture  and  are 
used  where  an  inconspicuous  scar  is  particularly  desirable, 
as  on  the  face.  Only  the  finer  grades  are  used  for  this 
purpose. 

Silver  Ti'/'/T.1  This  material  has  the  advantage  over  other 
suture  materials  of  having  a  germicidal  or  at  least  a  restrain- 
ing influence  on  bacteria.  If  we  remember  that  absolute 
sterilization  of  the  skin  is  not  possible  by  any  means,  we 
must  see  that  in  silver  wire  as  a  skin  suture  we  have  a  safe 
and  valuable  material.  Recent  annealing  by  heating  to  a 
dull  red  increases  the  flexibility  of  the  wire  but  almost 
totally  destroys  its  germicidal  property.  This  will  reappear 
in  a  month  and  is  not  disturbed  by  boiling.  Therefore  pre- 
pare it  by  boiling  for  ten  minutes  in  the  I  per  cent,  soda 
solution. 

Sponges.  The  best  absorbents  to  use  in  surgical  work  are 
those  whose  sterility  is  undoubted.  Pads  of  gauze  are  easily 
sterilized  by  steam  as  for  dressings.  Sea  sponges2  may  be 
prepared  by  beating  with  a  wooden  mallet  to  remove  sand 
and  dirt.  Soak  in  a  1-64  solution  of  hydrochloric  acid  for 

1  Bolton,   Transactions  Association  American  Physicians,   1894. 
"  McBurney,    "International   Text-Book   of   Surgery/'   June,    1900,   p. 
284. 


PREPARATION    OF    INSTRUMENTS,    ETC.  219 

twelve  hours  to  remove  lime  deposits.  Wash  in  running 
warm  water.  Soak  for  fifteen  minutes  in  a  saturated  solu- 
tion of  permanganate  of  potassium,  then  place  in  a  satu- 
rated solution  of  oxalic  acid  until  they  are  perfectly  bleached. 
After  immersion  for  half  an  hour  in  this  solution  rinse 
thoroughly  in  sterile  water  and  put  in  a  i-iooo  bichloride 
of  mercury  solution  for  twenty-four  hours.  Remove  and 
place  in  1-20  carbolic  acid  solution  until  required  for  use. 
At  operation  remove  from  solution,  rinse  out  in  normal  salt 
solution,  and  place  in  receptacle  filled  with  salt  solution.  If 
sea  sponges  are  used  on  a  septic  case  they  should  be  thrown 
away  and  no  attempt  made  to  resterilize  them.  If  used  on 
clean  cases  they  may  be  resterilized  as  above. 

Dressings.  The  two  materials  universally  used  to  dress 
wounds  are  "  gauze  "  or  cheese-cloth  and  absorbent  cotton. 
If  they  are  properly  sterilized,  the  impregnation  of  gauze 
or  cotton  with  antiseptics  does  not*  add  to  their  value. 
Gauze  is  usually  cut  in  portions  one  yard  square  and  folded 
in  pieces  called  compresses.  A  number  of  compresses  are 
wrapped  with  a  piece  of  cotton  cloth  and  the  edges  stitched 
loosely  into  a  closed  bundle.  After  sterilization  by  the  frac- 
tional method,  the  bundles  can  be  placed  in  sterile  jars  or 
receptacles  and  each  bundle  removed  as  needed.  Ripping 
open  the  stitches  gives  untouched  sterile  bundles  of  com- 
presses, convenient  and  handy  for  using.  Cotton  is  sterilized 
by  the  fractional  method  in  rolls  or  bundles  as  for  gauze. 
These  dressings  should  be  warmed  before  being  placed  in 
the  steam  sterilizer  or  they  will  be  unnecessarily  wet  when 
removed. 

Irrigating  Solutions.  Chemical  germicides,  such  as  bichlo- 
ride of  mercury  when  in  solution,  cause  necrosis  of  tissue. 
Plain  sterile  water  causes  maceration  of  epithelium.  Nor- 
mal salt  solution  is  the  least  irritating  to  the  tissues  and  is 
the  one  most  generally  employed  for  irrigating  purposes.  It 

T9 


22O  MANUAL    OF    BACTERIOLOGY. 

is  .6  per  cent,  sodium  chloride,  prepared  roughly  by  adding 
a  teaspoonful  of  salt  to  the  pint  of  water.  This  solution 
may  be  sterilized  by  boiling  for  half  an  hour  on  three  con- 
secutive days.  It  does  not  injure  tissue,  and  may  be  freely 
used  in  operations  for  irrigating.  It  has  no  germicidal  or 
antiseptic  properties. 

Accident  wounds  are  generally  lacerated  or  contused  and  may  con- 
tain pathogenic  bacteria.  They  should  be  promptly  and  carefully 
cleansed  with  sterile  salt  solution,  wiped  with  sterile  gauze  and  if 
necessary  scrubbed  vigorously  with  sterile  soap  and  brush  to  remove 
all  infectious  dirt.  When  there  is  any  doubt  of  this  being  accomplished 
it  is  better  to  dress  such  wounds  wide  open,  filled  with  sterile  gauze, 
for  forty-eight  hours  'or  more.  Retained  blood  clots  form  a  good 
medium  for  the  development  of  bacteria  so  that  drainage  for  a  day  or 
two  is  safer  in  doubtful  cases. 

Infected  wounds.  There  is  no  known  method  for  promptly  sterilizing 
infected  wounds  without  destroying  tissue.  An  infected  wound,  if 
the  infection  be  not  too  deep,  may  be  sterilized  by  cauterizing  with  pure 
carbolic  acid. 

Care  must  be  exercised  in  the  application  of  antiseptic  solutions  in 
infected  wounds  for  the  antiseptic  rarely  penetrates  as  deeply  into  the 
tissues  as  the  bacteria  are  found,  therefore,  further  necrosis  of  tissue 
and  mechanical  cleanliness  are  about  all  they  accomplish. 

After-treatment  of  wounds.  Close  attention  to  details  is  important  in 
the  technique  of  a  first  dressing  after  an  operation.  All  instruments, 
irrigating  fluids,  bowls,  basins,  etc.,  are  to  be  sterilized.  When  the 
dressing  is  removed  the  skin  surrounding  the  wound  should  be  cleansed 
by  washing  with  salt  solution  or  peroxide  of  hydrogen.  The  sutures 
or  drainage  should  be  removed  with  sterile  forceps,  and  fresh  sterile 
dressings  applied.  If  a  wound  is  found  infected,  all  accumulations  of 
blood-clot,  pus,  etc.,  should  be  gently  and  carefully  washed  out,  and 
free  drainage  provided.  In  the  care  of  infected  wounds  careful  attention 
must  be  paid  to  maintaining  mechanical  cleanliness  and  avoiding 
infection  with  some  organism  which  may  not  be  already  present. 

It  should  be  borne  in  mind  that  anything  that  tends  to  depress  a 
patient's  resisting  powers  encourages  infection ;  such  as  prolonged 
exposure  to  cold  during  an  operation,  loss  of  blood  and  infliction  of  a 
great  degree  of  surgical  shock. 


PART    III. 


NON-PATHOGENIC    BACTERIA. 

THE  number  of  varieties  of  non-pathogenic  bacteria  is 
very  large.  Eisenberg1  describes  376  species  of  bacteria, 
mostly  non-pathogenic.  Sternberg2  enumerates  489  species, 
including  the  pathogenic  varieties,  but  the  majority,  of 
course,  are  non-pathogenic.  Fliigge3  considers  about  500 
species  of  bacteria.  Migula4  recognizes  nearly  1,300,  and 
Chester5  about  800  species.  Probably  some  of  the  bacteria 
which  have  been  described  as  distinct  species  are  in  reality 
not  different;  but,  on  the  other  hand,  it  is  also  probable 
that  a  still  larger  number  of  species  have  not  been  described 
at  all ;  how  many  it  is  impossible  to  say.  In  a  work  of  this 
character  it  is  feasible  to  mention  only  a  few  of  the  com- 
monest and  best-known  species  of  non-pathogenic  bacteria. 

Micrococcus  agilis. — Found  in  water;  coccus  about  i  p 
in  diameter,  usually  appearing  as  diplococci,  sometimes  as 
streptococci  and  tetrads ;  liquefies  gelatin  slowly ;  grows  at 
room  temperature,  on  ordinary  culture-media,  forming  a 
rose-red  pigment  on  agar  and  potato.  This  micrococcus  is 
remarkable  in  being  actively  motile ;  it  possesses  a  flagellum. 
It  is  stained  by  Gram's  method. 

Micrococcus  urese. — Found  in  decomposed,  ammoniacal 
urine  and  in  the  air ;  coccus  .8  to  I  [*•  in  diameter,  occurring 

"  Bakteriologische  Diagnostik,"  1891. 

"  Manual  of  Bacteriology,"  1893. 
'"Die  Mikro  organism  en"  1896. 
1 "  System  der  Bakterien,"  1900. 
5  "  Manual  of  Determinative  Bacteriology,"   1901. 


222  MANUAL    OF    BACTERIOLOGY. 

singly  or  in  various  combinations ;  does  not  liquefy  gelatin ; 
facultative  anaerobic;  grows  rapidly,  best  at  30°  to  33°  C. ; 
grows  on  ordinary  gelatin,  but  best  on  special  media;  it 
decomposes  urea,  producing  ammonia  and  carbon  dioxide, 
which  form  ammonium  carbonate. 

Sarcinae. — There  is  a  large  number  of  species  of  sarcinse. 
They  are  common  organisms  in  the  air.  They  frequently 
contaminate  plate-cultures.  Many  of  the  sarcinae  of  the 
air  present,  in  cultures,  growths  having  brilliant  colors, 
from  which  some  of  them  are  named ;  thus  there  are  orange, 
yellow,  rose-colored  and  white  sarcinae,  and  others. 

Sarcina  pulmonum. — Found  in  the  air  passages  of  man; 
I  to  1.5  /J.  in  diameter,  occurring  in  tetrads  or  cubes  of  eight 
cells;  aerobic;  does  not  liquefy  gelatin;  grows  slowly,  best 
at  ordinary  temperatures,  preferably  upon  gelatin.  It  de- 
composes urine  with  the  formation  of  ammonia.  It  is  said 
to  form  endogenous  spores  which  are  extremely  resistant 
to  heat. 

Sarcina  ventriculi. — Found  in  the  stomachs  of  man  and 
of  animals;  2.5^  in  diameter,  occurring  in  cubes  of  eight 
cells  or  more;  it  does  not  liquefy  gelatin;  aerobic;  grows 
on  ordinary  culture-media;  the  growths  tend  to  become 
yellow.  Small  numbers  of  sarcinre  may  occur  in  the  normal 
human  stomach ;  the  presence  of  large  numbers  indicates  the 
existence  of  abnormal  fermentative  processes. 

Bacillus  fluorescens  liquefaciens. — Found  in  water  and 
putrid  fluids;  very  common;  appears  as  a  small  rod,  actively 
motile;  aerobic,  but  somewhat  variably;  liquefies  gelatin; 
grows  rapidly  at  ordinary  temperatures  upon  the  usual  cul- 
ture-media. It  forms  a  pigment  having  a  beautiful  greenish- 
yellow  fluorescence,  best  seen  in  transparent  media ;  the 
growth  on  potato  has  a  brown  color.  Does  not  stain  by 
Gram's  method  and  does  not  form  spores. 

Bacillus  fluorescens  putidus. — Found  in  water;  a  short 
rod  with  rounded  ends;  actively  motile;  does  not  liquefy 


NON-PATHOGENIC    BACTERIA.  223 

gelatin;  aerobic;  does  not  form  spores;  grows  rapidly  at 
l  lie  ordinary  temperatures  upon  the  common  media.  Gel- 
atin cultures  give  off  a  powerful,  foul  odor  of  trimethyl- 
amin.  It  produces  a  greenish,  fluorescent  pigment,  best 
seen  in  transparent  media;  on  potato  the  growths  form  a 
thin,  gray  to  brown,  slimy  layer. 

There  are  several  other  fluorescing  bacilli,  mostly  found 
in  water. 

Bacillus  Indicus. — Found  by  Koch  in  the  stomach-con- 
tents of  an  ape  in  India ;  a  fine  short  bacillus  with  rounded 
ends;  motile;  does  not  form  spores;  facultative  anaerobic; 
liquefies  gelatin;  grows  rapidly,  best  at  35°  C.  upon  the 
ordinary  media ;  produces  a  brick-red  pigment.  Very  large 
doses  injected  into  rabbits  caused  death  in  three  to  twenty- 
four  hours. 

Bacillus  prodigiosus. — \Yiclely  disseminated  in  the  at- 
mosphere of  certain  places ;  a  short  bacillus  with  rounded 
ends,  in  form  often  nearly  like  the  micrococci ;  facultative 
anaerobic;  not  motile,  as  a  rule;  does  not  form  spores; 
liquefies  gelatin  rapidly;  grows  rapidly,  best  at  25°  C.  on 
the  ordinary  culture-media;  milk  is  coagulated;  gas  forms 
in  sugar-media :  cultures  on  potatoes  give  off  a  foul  odor 
of  trimethylamin.  A  brilliant  red  color,  which  only  de- 
velops in  the  presence  of  oxygen,  appears  in  cultures.  The 
pigment  appears  as  granules  outside  of  the  bacteria. 

Bacillus  violaceus  (of  Berlin). — Found  in  water;  a  slim 
rod  with  rounded  ends  which  may  form  threads;  actively 
motile;  facultative  anaerobic;  liquefies  gelatin  rapidly; 
forms  endogenous  spores  placed  near  the  centers  of  the  ba- 
cilli ;  grows  rapidly,  and  not  at  high  temperatures,  upon 
ordinary  media,  forming  a  deep,  violet-colored  pigment. 
There  are  several  bacilli  related  to  this  one. 

Bacillus  amylobacter  (Clostridium  butyricum,  Bacillus 
butyricus,  Prazmowski). — Found  widely  distributed  in  na- 


224  MANUAL   OF    BACTERIOLOGY. 

ture  in  decomposing  vegetable  material  and  in  the  stomachs 
of  ruminant  animals;  a  large,  thick  rod  with  round  ends, 
often  arranged  in  chains;  actively  motile;  anaerobic;  forms 
spores,  which  are  located  in  the  center  of  the  bacillus  and 
give  it  a  spindle-shaped  form,  or  at  one  end  when  it  has  the 
outline  of  a  tadpole;  has  not  been  cultivated  satisfactorily 
on  ordinary  media;  grows  best  at  35°  to  40°  C. ;  decom- 
poses carbohydrates  with  the  formation  of  butyric  acid ; 
decomposes  cellulose.  Organisms  of  similar  form  have 
been  found  as  fossils  belonging  to  the  carboniferous  period. 

Bacillus  butyricus  (Hueppe). — Found  in  milk;  appears 
as  a  small,  irregular  rod,  also  forming  threads;  very  actively 
motile ;  aerobic ;  rapidly  liquefies  gelatin ;  forms  centrally 
located  spores;  grows  best  at  35°  to  40°  C. ;  grows  rapidly 
on  ordinary  media;  coagulates  milk,  redissolving  the  coag- 
ulum,  producing  also  butyric  acid.  A  large  number  of 
bacteria,  both  aerobic  and  anaerobic,  produce  butyric  acid 
fermentation. 

Bacillus  megatherium. — Obtained  by  DeBary  from 
cooked  cabbage-leaves ;  common  on  plants  and  earth ;  a  large 
bacillus  with  rounded  ends,  often  forming  chains;  motile; 
slowly  liquefies  gelatin ;  aerobic ;  forms  spores,  especially  in 
potato  cultures;  grows  rapidly  at  room  temperature  on  the 
ordinary  media. 

Bacillus  mesentericus  vulgatus  (Potato  bacillus).— 
Found  on  potatoes;  common  in  earth;  a  large,  long  rod 
with  rounded  ends,  often  forming  long  chains ;  motile ;  it 
is  stained  by  Gram's  method ;  liquefies  gelatin ;  aerobic ; 
forms  spores;  grows  rapidly,  best  at  about  20°  C. ;  grows 
on  ordinary  media,  forming  on  potato  a  thin,  wrinkled 
membrane  which  spreads  rapidly  over  the  surface.  It 
coagulates  milk,  redissolving  the  coagulmn.  It  possesses 
numerous  flagella.  The  spores  are  extremely  resistant  to 
heat. 


NON-PATHOGENIC    BACTERIA. 

Bacillus  phosphorescens  Indicus. — Obtained  from  sea- 
water;  a  small,  thick,  rod-shaped  bacillus  with  rounded 
ends,  also  forming  threads;  actively  motile;  not  stained 
by  Gram's  method;  liquefies  gelatin;  aerobic.  It  grows 
slowly,  best  between  20°  and  30°  C,  upon  the  usual  media; 
except  milk  and  potato.  Its  cultures,  when  old,  especially 
when  on  animal-  nutrient-media  and  in  the  presence  of 
certain  sodium  salts,  are  phosphorescent  in  the  dark. 

There  are  various  other  bacilli  which  produce  phospho- 
rescence, some  of  which  do  not  liquefy  gelatin. 

Bacillus  mycoides  (Bacillus  ramosus,  Wurzelbacillus). 
Found  in  the  earth  and  in  water,  very  common;  a  large, 
short  bacillus  with  rounded  ends,  often  forming  chains  and 
threads ;  slightly  motile ;  liquefies  gelatin ;  aerobic ;  forms 
centrally  located,  oval  spores;  gro\vs  rapidly  at  room  and 
incubator  temperatures  upon  the  usual  media.  It  is  said 
to  rapidly  decompose  albumin  with  the  formation  of 
ammonia. 

Bacillus  subtilis  (Hay  bacillus). — Found  on  hay,  in  the 
air,  water,  ground  and  decomposing  fluids;  very  common; 
a  large  bacillus  somewhat  resembling  the  anthrax  bacillus 
in  form,  with  rounded  ends,  often  forming  chains  or  long 
filaments;  motile;  possessing  flagella;  liquefies  gelatin; 
aerobic;  it  is  stained  by  Gram's  method.  It  may  have 
large,  centrally  located  spores,  which  form  best  on  potato 
at  about  30°  C.  The  spores  are  extremely  resistant  to  heat 
and  to  chemical  germicides.  It  grows  best  at  about  30°  C. 
upon  the  ordinary  culture-media;  milk  is  peptonized. 
Bacillus  subtilis  may  easily  be  isolated  in  pure  culture  by 
adding  finely-cut  hay  to  bouillon ;  place  in  the  steam  ster- 
ilizer for  five  or  ten  minutes;  then  let  the  tubes  develop  in 
the  incubator.  Plates  made  from  the  bouillon  will  probably 
show  colonies  of  the  bacillus  subtilis  only,  as  the  steam 
may  be  expected  to  have  destroyed  all  organisms  except 


226 


MANUAL    OF    BACTERIOLOGY. 


its  very  resistant  spores.     The  hay  bacillus  is  entirely  with- 
out pathogenic  properties. 

FIG.  49. 


Bacillus   subtilis.       (X  1000.) 

Bacillus  erythrosporus. — Found  in  decomposing  fluids 
and  water;  a  slim  bacillus  with  rounded  ends;  motile;  does 
not  liquefy  gelatin;  facultative  anaerobic;  forms  oval,  red- 
colored  spores,  two  to  eight  in  each  filament ;  grows  rapidly, 
only  at  ordinary  temperatures;  produces  a  greenish-yellow 
fluorescent  pigment.  On  potato  it  forms  a  limited,  reddish 
growth,  becoming  nut-brown. 

Bacillus  cyanogenus  (Bacterium  syncyanum,  Bacillus 
lactis  cyanogenus,  Bacillus  of  blue  milk). — A  bacillus  of 
variable  size,  with  rounded  ends;  motile;  spore  formation 
doubtful;  is  aerobic;  not  stained  by  Gram's  method;  grows 
rapidly  at  ordinary  but  not  so  well  as  incubator  tempera- 
tures on  the  usual  culture-media ;  does  not  liquefy  gelatin  ; 
produces  a  grayish-blue  pigment,  brighter  in  acid  media, 
at  ordinary  temperatures;  milk  is  not  coagulated,  or  ren- 
dered acid. 


NON-PATHOGENIC    BACTERIA.  22/ 

Bacillus  acidi  lactici  (Hueppe). — Found  in  sour  milk;  a 
short,  plump  rod;  not  motile;  does  not  liquefy  gelatin; 
facultative  anaerobic;  grows  on  the  ordinary  media;  in 
milk  causes  development  of  lactic  acid  with  precipitation 
of  casein  and  production  of  gas  and  alcohol.  It  belongs  in 
the  same  group  as  B.  coli  communis  and  B.  lactis  aerogenes 
(see  Part  IV.). 

There  are  numerous  other  bacteria,  such  as  the  bacte- 
rium acidi  lactici,  which  cause  the  formation  of  lactic  acid 
in  milk. 

Bacterium  ureae. — A  short,  thick  bacillus  with  rounded 
ends;  net  motile;  aerobic;  found  in  ammoniacal  urine; 
grows  slowly  at  room  temperature  upon  gelatin,  which  is 
not  liquefied ;  decomposes  urea,  forms  ammonium  carbonate. 

Bacterium  Zopfii. — Found  in  the  intestines  of  hens,  in 
water  and  in  fecal  matter;  a  bacillus  .75  to  i  JJL  broad  and  2 
to  5  /j.  long ;  may  form  threads.  Actively  motile ;  does  not 
liquefy  gelatin;  aerobic;  involution  forms  are  often  seen 
and  they  have  been  described  as  spores;  grows  rapidly, 
best  at  20°  C.  upon  gelatin;  forms  branching  zoogloese. 
It  is  a  member  of  the  same  group  as  B.  proteus  (see  Part 
IV.)- 

Spirillum  rubrutn. — Found  by  Esmarch  in  the  putrefy- 
ing cadaver  of  a  mouse;  short  spirals  twice  the  breadth  of 
the  cholera  spirillum,  usually  with  one  to  three  turns;  in 
bouillon  growing  into  long  spirals ;  motile  with  flagella ; 
spore  formation  doubtful ;  facultative  anaerobic ;  does  not 
liquefy  gelatin;  grows  slowly,  best  at  about  37°  C.  on  the 
ordinary  media;  produces  a  wine-red  pigment  only  when 
the  air  is  excluded. 

Spirillum  or  Spirochaeta  dentium. — Found  in  the  mouths 
of  healthy  persons,  on  the  margins  of  the  gums  when  they 
are  covered  with  a  dirty  deposit;  long  spirals  with  several 
windings  uneven  in  thickness ;  has  not  been  cultivated. 

20 


^28  MANUAL    OF    BACTERIOLOGY. 

Spirillum  sputigenum. — Found  in  the  human  mouth  in 
healthy  persons  at  the  margin  of  the  gums;  curved  rods  or 
short  spirals  which  resemble  the  spirillum  of  cholera  in 
form ;  has  not  been  cultivated. 

Spirillum  rugula  (Vibrio  rugula). — Found  in  swamp 
water,  in  fecal  matter,  and  in  the  tartar  of  the  teeth ;  a 
curved  rod  .5  to  2.5^  broad  and  6  to  8/2  long,  having  one 
flat  spiral  winding;  motile,  with  flagella  at  the  ends;  prob- 
ably anaerobic;  forms  spores  located  at  the  ends. 


FIG.  50. 


- 


^  X 

.  <\ 


Spirilla  from  Swamp  Water.      (X  about  500.) 

Spirillum  volutans. — Found  in  swamp  water;  very  long 
spirals  with  several  turns;  1.5  to  2  /*  broad  and  25  to  30  // 
long;  motile,  with  a  flagellum  at  each  extremity.  The 
protoplasm  is  granular. 

Spirillum  undula. — Found  in  putrefying  infusions  con- 
taining organic  matter ;  a  rather  short  spiral  form  with  three 
turns  or  less,  about  i  [*  thick  and  8  to  12^  long;  actively 
motile,  with  a  tuft  of  flagella  at  each  extremity;  has  been 
cultivated  on  agar. 


NON-PATHOGENIC    BACTERIA.  229 

Spirillum  or  Spirochaeta  plicatile. — Found  in  swamp 
water;  spiral  forms  of  various  lengths;  sometimes  100  to 
200 /•*  long;  actively  motile. 

The  spirilla  (vibrios  or  comma-shaped  forms),  closely 
resembling  the  spirillum  of  cholera,  will  be  considered  in 
connection  with  that  organism.  A  form  of  chronic  pseudo- 

FIG.  51. 


Spirilla    from    Swamp    Water,    showing   Flagella,    Loffler    Stain.      (X  1000.) 

membranous  inflammation  of  the  pharynx  has  been  attrib- 
uted to  an  organism  called  the  fusiform  bacillus  or  spirillum 
of  Vincent.1 

Higher  Bacteria. — Certain  organisms  (beggiatoa,  thio- 
thrix,  leptothrix,  cladothrix,  actinomyces  or  streptothrix) 
of  more  complicated  structure  than  most  bacteria,  but  re- 
sembling them  in  many  respects,  are  called  "  higher  bac- 
teria." They  consist  of  definite  filaments  which  are  usually 
made  up  of  rod-shaped  elements,  but  the  relation  between 
these  elements  is  very  intimate.  Some  of  them  (beggiatoa, 

1  Mayer,  American  Journal  Medical  Sciences,  Vol.  123,  1902,  p.  187. 


230  MANUAL    OF    BACTERIOLOGY. 

thiothrix)  contain  sulphur  granules.  Many  of  them  occur 
in  water.  There  are  forms  among  them  which  are  found 
attached  to  some  object  by  one  end  of  the  filament  (thio- 
thrix). Some  of  them  (actinomyces  or  streptothrix)  have 
branching  filaments,  which  are  rarely  seen  among  the  lower 
bacteria  (see  page  119).  Often  one  end  of  the  filament  be- 
comes specialized  for  the  purposes  of  reproduction.  The 
fungus  of  actinomycosis  is  the  best  known  of  this  group. 
There  are  many  other  members,  however,  both  pathogenic 
and  non-pathogenic.  Most  of  them  require  still  further 
study.  The  tubercle  bacillus  and  other  acid-proof  bacilli 
which  resemble  it,  have  some  points  of  resemblance  with 
actinomyces  (see  B.  tuberculosis,  Part  IV.). 

Leptothrix  buccalis. — Found  in  the  mouth  cavity.  This 
name  has  been  applied  to  large,  twisted,  thread-like  organ- 
isms, in  which  segments  can  be  demonstrated  with  diffi- 
culty or  not  at  all.  Apparently,  different  organisms  have 
been  described  under  this  name.  Vignal  claims  to  have 
cultivated  a  leptothrix  buccalis.  Miller  recognizes  two 
principal  species,  neither  of  which  could  be  cultivated,— 
leptothrix  innominata,  which  shows  no  transverse  divisions, 
and  which  is  stained  faintly  yellow  by  iodine;  and  bacillus 
buccalis  maximus,  in  which  the  transverse  divisions  are 
distinct,  and  which  is  stained  brownish-violet  by  iodine. 
Miller's  leptothrix  maxima  buccalis  is  similar  to  the  last 
except  in  lacking  the  iodine  reaction. 

A  variety  of  leptothrix,  or  a  nearly  related  organism,  ap- 
pears to  be  the  most  frequent  cause  of  the  form  of  gangren- 
ous inflammation  of  the  mouth  and  genitals  called  noma. 
It  stains  faintly  by  Gram's  method.  It  does  not  grow  on 
ordinary  media.1  Another  organism  of  this  group  has  been 

1Blumer    and     MacFarlane,     American    Journal    Medical    Sciences, 
November,  1901. 


YEASTS    AND    MOULDS. 


231 


described   which   is  pathogenic   to  a  number  of   domestic 
animals.1 

Yeasts  and  Moulds. — In  the  course  of  bacteriological 
work  one  constantly  encounters  yeasts  and  moulds,  which, 
although  not  bacteria,  must  nevertheless  be  understood  and 
recognized  to  avoid  error.  Accidental  contamination  of 
tubes  or  plates  is  likely  to  be  the  result  of  the  growth  of  some 

FIG.  52. 


Yeast  Cells,  stained  with  Fuchsin.      (X  1000.) 

of  these  forms.  The  yeasts  generally  go  by  the  name  of 
sac  char  omyces,  of  which  there  are  several  species.  The 
saccharomyccs  cerevisia  is  the  ordinary  yeast  of  alcoholic 
fermentation.  Some  of  the  yeasts  present  colored  growths 
—red,  white  and  black.  They  consist  of  large,  oval  cells, 
which  readily  stain  with  the  aniline  dyes.  They  multiply  by 

1  It  has  also  been  called  "  Necrosis  bacillus,"  and  "  Streptothrix 
cuniculi."  Pearce,  University  of  Pennsylvania  Medical  Bulletin,  Novem- 
ber, 1902. 


232 


MANUAL    OF    BACTERIOLOGY. 
FIG.  S3- 


a.   Penicillium  glaucum.     b.  Oidium  lactis.     c.  Aspergillus  glaucus.     d.  The 
same  more  highly  magnified.      c.  Mucor  mucedo    (Baumgarten). 


r 

YEASTS    AND    MOULDS.  233 

the  protrusion  of  a  little  bud  from  the  cell,  which  develops 
into  a  new  cell.  In  an  actively  germinating  growth  of  yeast 
these  budding  cells  are  readily  distinguished  (Fig.  52). 

Yeasts  have  been  found  that  were  pathogenic  to  animals. 
They  have  also  been  supposed  to  be  the  cause  of  some 
malignant  tumors,  but  this  view  has  been,  for  the  most  part, 
abandoned. 

Among  the  moulds  the  varieties  most  commonly  en- 
countered are  the  mucor,  the  penicillium,  the  aspergillus 
and  the  oidlum.  There  are  various  species  of  each  of 
them.  They  consist  of  cells  arranged  end  to  end,  making 
a  thread-like  body  called  a  hypha.  The  threads  are  matted 
together  and  form  a  mycelium.  Certain  threads  project 
upward  from  the  mycelium,  and  on  them  are  borne  spores, 
or  conidia.  The  arrangement  of  the  spores  is  characteristic 
in  each  variety  of  mould  (Fig.  53).  A  group  of  organisms 
exist  which  have  affinities  both  with  yeasts  and  mould- 
fungi.  Some  of  them  are  pathogenic.  The  form  of  infec- 
tion of  the  mouth  called  thrush,  is  due  to  a  fungus  of  this 
class,  which  is  generally  considered  an  oidium.  A  chronic 
inflammatory  affection  of  the  skin  (blastomycetic  der- 
matitis) is  due  to  related  organisms.1  The  Sporotricha  of 
Schenck2  which  produces  chronic  subcutaneous  abscesses, 
may  be  mentioned  here,  provisionally.  A  number  of  skin 
affections,  such  as  Tinea  favosa  and  Tinea  trichophytina, 
are  due  to  fungi,  which  have  some  similarity  with  those 
above  mentioned. 

Among  the  mould  fungi,  several  species  of  aspergillus  and 
of  mucor  are  pathogenic.  Man,  as  well  as  the  lower  ani- 
mals, may  be  affected.  In  man  the  lungs  may  be  involved 
in  a  broncho-pneumonia  (pneumonomycosis),  usually  due  to 

'Ricketts,  Journal  of  Medical  Research,  Vol.   VI.,    1901;    Hyde  and 
Montgomery,  Journal  American  Medical  Association,  June  7,  1902. 
2Hektoen,  Journal  Experimental  Medicine,  Vol.  V. 


234  MANUAL    OF    BACTERIOLOGY. 

aspergillus,  and  often  secondary  to  some  preexisting  disease 
of  the  lung.  Mould  fungi,  especially  aspergillus,  may  grow 
in  the  external  ear  (otomycosis).  The  growth  is  usually 
superficial.  These  fungi  rarely  produce  lesions  in  other 
organs. 


PART    IV. 


PATHOGENIC  BACTERIA. 

Suppuration  and  Allied  Conditions. — The  occurrence  of 
suppuration  is  characterized  by  certain  appearances  which 
we  are  accustomed  to  describe  under  the  name  of  inflamma- 
tion. The  study  of  inflammation  belongs  to  pathology,  and 
cannot  be  considered  here.  However,  certain  evidences 
which  are  characteristic  of  the  suppurative  variety  of  inflam- 
mation need  to  be  outlined  on  account  of  their  relation  to  the 
action  of  the  pyogenic  bacteria. 

In  a  suppurating  area,  as  is  well  known,  the  blood-ves- 
sels are  dilated,  and  the  lymph-spaces  become  filled  with 
serum.  Leucocytes  are  attracted  to  the  neighborhood  in 
large  numbers,  we  may  suppose  by  a  positive  chemotaxis, 
and  crowd  the  small  veins  and  capillaries.  The  leucocytes, 
by  reason  of  their  amoeboid  movement,  pass  through  the 
walls  of  the  vessels  at  little  openings  filled  with  cement- 
substance,  situated  between  the  lining  endothelial  cells.  Ac- 
cording to  the  theory  of  phagocytosis,  they  are  bent  on  find- 
ing the  irritant  which  has  led  to  the  inflammation,  and  upon 
isolating  it  and  rendering  it  harmless.  At  the  point  which 
appears  to  be  the  center  of  the  inflammatory  area  there  is 
usually,  but  not  always,  a  necrosis  of  the  cells  of  the  tissue; 
this  constitutes  the  central  slough  or  the  familiar  core  of 
some  boils.  The  necrosis  is  to  be  attributed  to  poisons 
formed  by  the  micrococci.  In  sections  cut  through  such  an 
abscess  the  nuclei  of  the  central  necrotic  cells  fail  to  take 
the  nuclear  stain ;  the  necrotic  mass  does  not  stain,  or  takes 


236  MANUAL    OF    BACTERIOLOGY. 

the  dye  diffusely  and  irregularly,  and  it  exhibits  many  fine 
granules. 

We  find  the  cells  of  the  tissues  surrounding  the  necrotic 
area  mingled  with  large  numbers  of  polynuclear  leucocytes, 
which  enclose  the  area  of  irritation. 

The  nuclei  of  the  cells  near  the  center  of  the  abscess  are 
frequently  broken  up  into  a  number  of  small  parts  (frag- 
mentation), which  indicates  the  commencement  of  their 
destruction.  In  sections  through  small  abscesses  it  is  pos- 
sible, by  means  of  a  double  stain  of  carmine  followed 
with  gentian-violet,  according  to  Gram's  method,  to  bring 
out  the  histological  character  of  the  tissue,  and  at  the  same 
time  to  stain  the  common  pyogenic  bacteria,  which  are 
usually  found  near  the  center  of  the  abscess  in  large  num- 
bers, even  making  masses  visible  with  a  low  power  of  the 
microscope.  Preparations,  most  convincing  and  of  great 
beauty,  may  be  secured  in  this  manner.  It  is  often  pos- 
sible to  demonstrate  masses  of  micrococci  filling  up  the 
lumina  of  capillaries  in  which  they  are  lodged  as  emboli. 

The  production  of  pus  in  the  center  of  the  abscess  is  due 
to  the  liquefaction  of  the  necrotic  tissue,  which  apparently 
results  from  the  action  of  some  peptonizing  ferment.  In 
the  liquid  thus  formed,  immense  numbers  of  the  polynu- 
clear leucocytes  are  found  floating,  and  they  constitute  the 
greater  part  of  the  so-called  pus-cells.  The  nuclei  of 
these  cells  are  obscured  by  clouds  of  extremely  fine  granules. 
The  granules  are  of  an  albuminoid  nature,  and  are  dissolved 
by  acetic  acid,  when  the  nuclei  become  visible.  The  nuclei 
generally  consist  of  three,  four,  five  or  more  portions. 
The  presence  of  the  fine  albuminoid  granules  in  the  pus- 
cells  is  to  be  counted  as  a  degenerative  change.  Although 
it  is  possible  to  produce  suppuration  in  laboratory  experi- 
ments by  the  introduction  of  sterilized  irritants,  such  as 
croton  oil,  in  the  vast  majority  of  cases  suppuration  is  due 
to  the  action  of  pyogenic  bacteria. 


PATHOGENIC    BACTERIA.  237 

Specimens  of  pus  will  nearly  always  be  found  to  contain 
bacteria,  which  can  be  demonstrated  by  cultivation,  and, 
as  a  rule,  also  in  smears  made  and  stained  upon  cover- 
glasses.  The  bacteria  are  generally  found  outside  the  pus- 
cells.  In  the  case  of  the  gonococcus  and  the  diplococcus 
intracellularis  meningitidis  they  are  characteristically  found 
in  pairs,  inside. of,  or  at  least  attached  to  the  pus-cells. 
The  character  of  the  suppuration  differs  somewhat  with  the 
different  species  of  pyogenic  bacteria.  The  kind  of  abscess 
above  described — localized  and  having  a  central  slough, 
usually  rather  slow  in  progress — is  typical  for  the  staphylo- 
coccus  pyogenes  aureus,  which  is  prone  to  produce  circum- 
scribed areas  of  suppuration.  The  streptococcus  pyogenes, 
on  the  other  hand,  oftener  leads  to  suppuration  of  a  more 
diffused  character,  such  as  we  see  in  cellulitis  and  erysipelas; 
but  either  organism  may,  at  times,  produce  the  effects 
usually  characteristic  of  the  other.  Pus  having  a  blue  or 
green  tinge  generally  owes  the  color  to  the  presence  of  the 
bacillus  pyocyaneus.  The  commonest  pus-producing  organ- 
ism is  then  the  Staphylococcus  pyogenes  aurcus,  and  next  to 
that  the  streptococcus  pyogenes.  Among  the  other  pyogenic 
bacteria  the  following  may  be  named  : 

Staphylococcus  pyogenes  albus,  including  staphylococ- 
cus  epidermidis  albus;  streptococcus  of  erysipelas  (prob- 
ably identical  with  streptococcus  pyogenes);  gonococcus; 
diplococcus  intracellularis  meningitidis ;  Staphylococcus 
pyogenes  ciireus;  micrococcus  tetragenus ;  micrococcus 
pyogenes  tennis,  which  may  be  the  same  as  the  micrococcus 
lanceolatus;  Staphylococcus  cereus  albus  and  flavus. 

Pus-formation  may  also  be  due  to  micrococcus  lanceola- 
tus, bacillus  pyocyaneus,  bacillus  proteus,  bacillus  coli 
communis,  bacillus  pyogenes  fetidus,  bacillus  pneumonias 
(of  Friedlander),  bacillus  aerogenes  capsulatus,  the  ray 
fungus  of  actinomycosis,  and  possibly  the  bacillus  of  bu- 


238  MANUAL    OF    BACTERIOLOGY. 

bonic  plague.  Besides  these  organisms,  there  are  others 
whose  effects  are  usually  more  marked  in  a  specific  way 
which  sometimes  form  pus,  as  the  bacilli  of  diphtheria, 
tuberculosis,  glanders  and  typhoid  fever. 

Frequently  two  or  more  species  of  pyogenic  bacteria  will 
be  found  associated. 

The  table  on  page  239,  quoted  from  Dowd,  shows  the  fre- 
quency of  the  occurrence  of  various  pyogenic  bacteria  in 
135  cases  of  different  types  of  suppuration. 

The  condition  of  the  animal's  tissues  is  of  great  impor- 
tance in  determining  whether  or  not  suppuration  is  to  oc- 
cur. It  will  be  seen  that  we  are  repeatedly  subjected  to 
infection  with  pyogenic  bacteria,  but  that  in  most  cases 
suppuration  nevertheless  does  not  occur.  The  local  condi- 
tions have  an  important  influence  in  determining  infection. 
Regions  of  hyperemia,  edema,  anemia  or  necrosis  are 
especially  liable  to  suppuration,  as  are  tissues  which  have 
been  bruised,  lacerated,  strangulated  or  otherwise  dam- 
aged. Furthermore,  the  general  condition  of  the  patient 
is  of  great  importance.  Chronic  diseases  and  conditions 
of  exhaustion  or  depression  dispose  to  suppuration,  and 
the  depraved  condition  of  the  tissues  in  diabetes  renders 
the  sufferer  from  this  disease  especially  liable  to  it.  These 
facts  have  already  been  enumerated  in  a  previous  chapter 
(page  165).  In  the  lower  animals  we  find  that  it  is  often 
very  difficult  to  produce  suppuration  artificially  with  the 
ordinary  pyogenic  bacteria.  In  rabbits  the  subcutaneous 
introduction  of  staphylococcus  pyogenes  aureus  frequently 
fails  to  produce  an  abscess.  Suppuration  is  likely  to  result, 
however,  if  an  irritant  body  like  a  piece  of  sterilized  potato 
or  sterilized  glass  be  introduced  along  with  the  bacteria. 

Pyogenic  bacteria  are  most  frequently  introduced  into 
the  body  through  the  agency  of  injuries  and  wounds  of 
various  sorts.  They  are  very  widely  disseminated  in 


PATHOGENIC    BACTERIA. 


239 


nature,  and  are  always  liable  to  be  clinging  to  external 
objects,  especially  in  cities  and  around  dwellings.  The 
infection  of  a  wound  in  this  manner,  when  the  suppuration 
is  of  a  spreading  character,  such  as  is  most  character- 
istic of  streptococcus  infection,  is  known  in  every-day  Ian- 


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C 

1 

Streptococcus  pyo^enes  alone 

Streptococcus  pyogenes  predominant.  .  .  . 

9 
23 

3 

7 

8 

Streptococcus  pyogenes   relatively  few.. 
Staphylococcus  pyogenes  aureus  alone.  . 

3 
ii 

I 
I 

6 
i 

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7 

i 
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Staphylococcus     pyogenes     aureus     pre- 

dominant 

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Staphylococcus    pyogenes    aureus     rela- 

tively few   

T  ? 

2 

Staphylococcus  pyogenes  or  epidermidis 

albus  alone   

I 

4 

2 

4 

2 

Staphylococcus  pyogenes  or  epidermidis 

albus  predominant 

.. 

Staphylococcus  pyogenes  or  epidermidis 

albus  relatively  few  

10 

c 

- 

6 

Staphylococcus  cereus  albus 

•j 

I 

2 

i 

Staphylococcus   citreus 

i 

2 

I 

No  growths  on  agar 

1  1 

Very  few  growths  on  agar  

7, 

7 

Bacillus  pyocyaneus   ... 

I 

7 

Bacillus  coli  communis 

7 

Overgrown 

4. 

2 

I 

Few  undetermined  colonies  

12 

2 

5 

5 

guage  as  "  blood-poisoning."  It  is  possible  for  infec- 
tion to  take  place  around  hair-follicles  through  the  un- 
broken skin.  In  such  instances  the  suppurative  inflamma- 
tion first  shows  itself  in  a  minute  red  pimple  with  a  hair  in 
the  center.  The  pimple  presently  becomes  a  pustule.  The 
process  may  cease  at  this  point,  or  it  may  be  only  the  com- 
mencement of  a  large  carbuncle  with  a  central  slough.  Such 
infection  has  been  produced  experimentally  on  the  human 


24O  MANUAL    OF    BACTERIOLOGY. 

skin  by  rubbing  in  cultures  of  staphylococcus  pyogenes 
aureus.  It  is,  furthermore,  the  constant  experience  of  post- 
mortem examiners  that  infection  may  occur  around  the  hair- 
follicles  when  no  wound  of  the  skin  has  been  inflicted. 

In  many  instances,  infection  with  the  pyogenic  bacteria 
follows  upon  some  preexisting  infection;  this  happens,  for 
instance,  in  tuberculosis,  when  tuberculous  lungs  become 
infected  with  streptococcus  pyogenes,  leading  to  the  forma- 
tion of  a  cavity.  It  is  a  common  occurrence  in  gonorrhea, 
after  the  acute  stage  of  the  disease  has  passed,  when  we 
find  the  gonococcus  in  the  pus,  mingled  with  other  pyo- 
genic micrococci.  Secondary  infection  with  pyogenic  bac- 
teria is  frequently  due  to  the  streptococcus  pyogenes,  often 
also  to  the  micrococcus  lanceolatus. 

Sometimes  we  are  obliged  to  admit  that  the  manner  in 
which  the  pyogenic  bacteria  enter  the  body  is  unknown. 

The  severe  general  symptoms,  familiar  to  every  physi- 
cian, often  accompanying  acute  suppuration,  indicate  the 
formation  of  toxic  bacterial  products  and  their  absorption. 
Experimental  evidence  of  the  formation  of  such  toxic  prod- 
ucts is  not  so  clear,  however,  for  the  pyogenic  organisms 
as  for  some  of  the  other  bacteria.  It  has  been  shown  that 
cultures  of  staphylococcus  pyogenes  aureus,  in  which  the 
bacteria  have  been  killed,  are  capable  of  producing  suppura- 
tion in  the  lower  animals. 

The  pyogenic  bacteria  play  a  somewhat  different  part  in 
producing  disease,  which  is  fully  as  important  as  the  typ- 
ical suppuration  seen  in  an  abscess.  This  happens  when 
the  suppurative  condition  is  mixed  with  other  phenomena, 
or  when  there  is  inflammation  of  another  variety  without 
suppuration  at  all ;  or  there  may  be  lesions  not  inflamma- 
tory in  a  strict  sense.  These  differences  in  their  action 
depend  largely  upon  the  organ  affected.  One  such  condi- 
tion is  osteomyelitis,  which  is,  usually,  suppuration  occur- 


I 

PATHOGENIC    BACTERIA.  24! 

ring  in  bone,  but  which  does  not  present  the  ordinary 
picture  of  pus-formation  owing  to  the  hard  and  unyielding 
character  of  the  tissue.  Other  conditions  of  very  great 
importance  are  meningitis,  pericarditis,  pleuritis,  pneumo- 
nia (croupous  and  broncho-),  peritonitis  and  endocarditis. 
It  will  be  observed  that  these  affections  are,  for  the  most 
part,  inflammations  of  the  serous  membranes.  Such  in- 
flammations, when  they  are  produced  by  pyogenic  bac- 
teria, are  likely  to  be  of  great  severity,  accompanied  by 
the  formation  of  fibrinous  exudates ;  pus-formation  may  or 
may  not  be  present.  We  find  that  the  cause  at  times  is 
the  staphylococcus  pyogenes  aureus;  this  is  often  the  case 
in  malignant  endocarditis.  Generally  speaking,  in  such  in- 
flammations the  streptococcus  pyogenes,  the  staphylococcus 
pyogenes  aureus,  and  the  pneumococcus  occur  most  com- 
monly, although  they  are  by  no  means  the  only  organisms 
found.  Many  cases  of  peritonitis  show  the  presence  of  B. 
coli  communis,  either  in  combination  with  other  bacteria, 
or  alone.1  This  is  explained  by  the  proximity  of  the  intes- 
tine, and  especially  by  the  frequent  occurrence  of  peritonitis 
after  perforation  of  the  intestine. 

In  inflammations  of  mucous  membranes  the  common 
pyogenic  organisms  play  the  most  important  though  not  an 
exclusive  part.  In  acute  bronchitis,  pneumococci  and  strep- 
tococci were  found  by  Ritchie  to  be  the  commonest  causes. 

In  inflammations  of  the  middle  ear  the  principal  causes  are 
the  pneumococcus,  the  streptococcus,  and  the  staphylococcus 
aureus  and  albus.3 

In  25  cases  of  acute  cystitis  in  women  Brown4  found 
B.  coli  communis,  15  times;  S.  pyogenes  albus,  5  times;  S. 

1  Flexner,  "  Etiology,  etc.,  of  Peritonitis,"  Philadelphia  Medical  Jour- 
nal, November  12,  1898. 

2  Ritchie,  Journal  Pathology  and  Bacteriology,  Vol.  VII.,  December, 
1900. 

3Hasslauer,  Ccntralblatt  f.  Baktcriologic,  XXXII.,  Ref.,  1902,  p.   174. 
Compare  Ibidem,  pp.  240  and  246. 

4  Johns  Hopkins  Hospital  Reports,  Vol.  X.,  1902. 


242  MANUAL    OF    BACTERIOLOGY. 

pyogenes  aureus,  2  times;  B.  typhosus,  I  time;  B.  pyocy- 
aneus,  i  time ;  B.  proteus  vulgaris,  i  time. 

A  number  of  investigators  have  recovered  from  cases  of 
acute  articular  rheumatism  organisms  resembling  the  pyo- 
genic  cocci.  Most  frequently  a  diplococcus  or  short  strepto- 
coccus has  been  found,  which  has  sometimes  produced 
arthritis  and  endocarditis  when  inoculated  into  rabbits. 


Staphylococcus  pyogenes  aureus  in  Pus,  stained  by  Gram's  Method. 
(X  1000.) 

From  a  point  where  there  is  suppuration  or  other  local- 
ized infection,  pyogenic  bacteria  may  enter  the  circulation 
and  become  widely  disseminated  throughout  the  body. 
That  happens  very  commonly  in  malignant  endocarditis.  In 
this  manner  secondary  or  metastatic  abscesses  may  be  pro- 
duced in  the  most  diverse  organs. 

The  term  pyemia  is  used  to  describe  the  dissemination  of 
pyogenic  bacteria  in  the  circulating  blood,  with  the  forma- 
tion of  metastatic  abscesses. 


PATHOGENIC    BACTERIA. 


243 


Staphylococcus  pyogenes  aureus.  — A  micrococcus  of 
variable  size,  arranged  in  irregular  clumps,  sometimes  in 
pairs;  about  .8  to  .9^  in  diameter;  not  motile  (Fig.  55).  It 
stains  by  Gram's  method;  it  is  facultative  anaerobic;  grows 
rapidly,  best  at  30°  to  37°  C.  It  liquefies  gelatin.  Upon 
gelatin  plates  small  colonies  appear  at  the  end  of  about  two 
days.  It  grows  well  upon  all  the  culture-media.  Milk  is 
coagulated.  It  does  not  lead  to  fermentation  with  the  pro- 
duction of  gas  but  produces  various  acids. 

FIG.  55. 


Staphylococcus  pyogenes  aureus,  Pure  Culture.      (X  1000.) 

The  growths  in  the  first  place  are  pale,  subsequently 
becoming  golden-yellow  in  color,  but  only  in  the  presence 
of  oxygen.  This  color  appears  well  on  all  media,  and  is 
especially  distinct  on  potato.  Sometimes  the  color  is  slow 
in  developing. 

In  a  fresh,  moist  condition  the  organism  is  killed  by  ten 
minutes'  exposure  to  58°  C. ;  in  a  desiccated  condition  it 
requires  a  temperature  of  90°  to  100°  C.  to  destroy  it.  It 


21 


244  MANUAL    OF    BACTERIOLOGY. 

resists  drying  in  a  considerable  degree.  In  thq  same  speci- 
men the  micrococci  may  have  quite  different  resisting  powers 
to  chemical  germicides.  Some  of  them  are  destroyed  by 
i-iooo  solution  of  bichloride  of  mercury  in  five  minutes; 
others  survive  exposure  to  the  same  for  from  ten  to  thirty 
minutes.  (Abbott.) 

Sterilized  cultures  introduced  into  animals  may  produce 
local  suppuration.  The  toxic  substances  occur  in  the  bac- 
terial cells.1 

As  has  already  been  mentioned,  the  staphylococcus  pyo- 
genes  aureus  is  the  commonest  of  the  pyogenic  bacteria 
in  man.  It  has  been  obtained  from  a  great  variety  of 
sources,  and  appears  to  be  able  to  exist  as  a  saprophyte. 
It  has  been  found  on  the  skin,  in  the  mouth,  in  the  nasal 
and  pharyngeal  mucus,  and  also  in  the  alimentary  canal. 
It  has  furthermore  been  detected  in  the  air  and  in  dust.  It 
appears  to  find  the  conditions  necessary  for  its  existence 
in  the  vicinity  of  human  habitations. 

Cultures  of  the  staphylococcus  pyogenes  aureus  vary 
considerably  in  virulence.  These  variations  are  sometimes 
to  be  explained  through  cultivation  on  unfavorable  media 
or  repeated  transplantation  from  one  medium  to  another; 
but  at  times  the  diminished  virulence  is  due  to  unknown 
causes.  The  lower  animals  used  for  experiments  are  not 
as  readily  infected  as  man.  The  local  introduction  in  rab- 
bits or  guinea-pigs  of  a  part  of  a  culture  of  staphylococcus 
pyogenes  aureus  may  be  entirely  without  effect.  The  use 
of  a  very  large  dose,  or  the  addition  at  the  same  time  of 
some  kind  of  irritant,  may  produce  an  abscess.  Large 
amounts  of  cultures  in  bouillon  may  often  be  injected  into 
the  peritoneal  cavity  of  the  dog  without  effect,  when  the 
simultaneous  addition  of  a  piece  of  sterile  potato  or  an 
injury  to  the  gut  may  lead  to  fatal  peritonitis.  Introduc- 

1  See  also  Morse,  Journal  Experimental  Medicine,  Vol.  I.,  p.  613. 


PATHOGENIC    BACTERIA. 


245 


FIG.  56. 


tion  of  fluid  cultures  into  the  venous  circulation  of  the  rabbit 
generally  produces  metastatic  abscesses  in  the  kidneys,  the 
heart-muscle  and  the  voluntary  muscles,  and  causes  death. 

In  man  this  organism  produces  sup- 
puration of  a  localized  character,  such 
as  we  are  familiar  with  in  boils  and 
carbuncles.  It  has  been  shown  to  be 
the  usual  cause  of  infectious  osteo- 
myelitis. Osteomyelitis  has  been  pro- 
duced experimentally  in  rabbits  by  the 
injection  of  the  staphylococcus  pyo- 
genes  aureus,  both  with  and  without 
previous  injury  to  the  bone  of  the  ani- 
mal. Ulcerative  endocarditis  has  on 
numerous  occasions  been  shown  to  be 
due  to  this  organism.  It  has  been 
found  possible  to  produce  ulcerative 
endocarditis  experimentally  in  animals 
by  the  injection  of  the  staphylococcus 
pyogenes  aureus  when  the  valves  of  the 
heart  have  first  been  mechanically  in- 
jured. The  staphylococcus  pyogenes 
aureus  has  also  been  found  in  acute 
abscesses  of  the  lymph-nodes,  tonsils, 
parotid  gland,  and  mammary  gland, 

.    .     ,       ~r  1  Staphylococcus   py- 

m  suppurating  joint  affections  and  em-    ogenes  aureus,  Geia- 
pyema.     It    appears,    furthermore,    in    tin  Culture,  i  Week 
acute  inflammation  of  the  serous  mem- 
branes,— pleuritis,    pericarditis,    peritonitis, — although    less 
frequently  than  the  streptococcus  pyogenes. 

Staphylococcus  pyogenes  albus. — In  form  and  manner 
of  growth  this  organism  behaves  like  the  staphylococcus 
pyogenes  aureus,  with  the  exception  that  it  produces  no 
colored  growths  and  its  cultures  appear  white.  Its  patho- 
genic properties  are  less  marked,  and  it  is  a  less  frequent 


246  MANUAL    OF    BACTERIOLOGY. 

cause  of  suppuration  than  the  staphylococcus  pyogenes 
aureus.  It  has,  however,  been  found  in  acute  abscesses  on 
numerous  occasions. 

Staphylococcus  epidermidis  albus. — According  to  Welch, 
the  epidermis  of  man  contains  with  great  regularity  the 
organism  to  which  he  gave  the  above  name,  and  which  he 
considers  to  be  a  variety  of  staphylococcus  pyogenes  albus. 
It  grows,  liquefies  gelatin,  and  coagulates  milk  more  slowly 
than  the  ordinary  staphylococcus  pyogenes  albus.  It  is, 
furthermore,  possessed  of  less  marked  pus-producing  ten- 
dencies. Welch  found  it  impossible  to  sterilize  the  skin  so 
as  to  remove  this  micrococcus  from  it.  The  organism  is 
usually  innocuous.  It  has  been  found  in  healthy  wounds 
on  numerous  occasions.  It  is  capable  of  causing  trouble 
in  wounds  when  necrotic  or  strangulated  tissues  are  present, 
or  where  a  foreign  body  like  a  drainage-tube  has  been  left 
in  the  wound.  It  is  a  common  cause  of  stitch  abscesses. 

Streptococcus  pyogenes. — Appears  as  micrococci  ar- 
ranged in  chains,  often  in  pairs,  when  the  adjacent  cocci 
may  be  flattened.  Sometimes  the  chains  are  very  long. 
The  diameters  of  the  cocci  vary  from  .4  to  i  //.  Attempts 
have  been  made  to  create  varieties  of  streptococci  accord- 
ing to  the  length  of  the  chains.  On  that  basis  a  strepto- 
coccus brevis  and  a  streptococcus  longus  have  been  de- 
scribed. 

The  streptococcus  pyogenes  is  not  motile.  It  stains  by 
Gram's  method.  By  the  method  of  Hiss  (page  57)  capsules 
may  sometimes  be  demonstrated.  It  is  facultative  anaero- 
bic; grows  best  in  the  incubator;  more  slowly  at  room  tem- 
perature, and  does  not  liquefy  gelatin.  In  gelatin  plates  it 
produces  small,  round,  white,  punctiform  colonies  which  are 
slow  of  development,  and  are  only  visible  after  about  three 
days.  It  grows  on  the  ordinary  media;  with  the  exception 
of  potato,  according  to  some  authors.  Milk  may  or  may  not 


f 

PATHOGENIC    BACTERIA.  247 

be  coagulated.     The  growths  are  never  very  luxuriant,  and 
may  die  out  entirely  after  a  few  transplantations. 

It  is  killed  by  exposure  to  52°  to  54°  C,  in  ten  minutes. 
The  streptococcus  pyogenes  occurs  frequently  on  the  mu- 
cous surfaces  of  the  healthy  body.  It  is  often  found  in 
pus,  especially  pus  of  spreading  inflammations  of  the  kind 

FIG.  57. 


Streptococcus  pyogenes,  from  a  Pure  Culture.      (X  1000.) 

known  as  cellulitis.  This  organism  is  the  commonest  infec- 
tious agent  in  puerperal  fever,  metritis  and  peritonitis.  It 
occurs  commonly  in  inflammations  of  the  serous  membranes 
— pleuritis,  pericarditis  and  peritonitis.  It  has  been  dis- 
covered many  times  in  ulcerative  endocarditis,  and  in 
broncho-pneumonia.  It  is  frequently  present  in  the  false 
membrane  found  in  genuine  diphtheria.  It  is  also  the  cause 
of  many  of  the  pseudo-membranous  or  so-called  "  diph- 
theritic "  affections  of  the  throat  where  the  Klebs-Loffler 
bacillus  of  diphtheria  is  wanting.  These  cases  may  be  in- 
distinguishable clinically  from  genuine  diphtheria,  and  their 


248  MANUAL    OF    BACTERIOLOGY. 

nature  will  only  be  revealed  on  bacteriological  examination. 
They  are,  however,  as  a  rule,  milder  than  genuine  diph- 
theria. The  pseudo-membranous  affections  of  the  throat 
which  occur  in  scarlet  fever  and  measles  are  generally  caused 

FIG.  58. 


• 


Streptococcus  pyogenes  in  Pus,  Gram's  Stain.      (X  1000.) 

by  the  streptococcus  pyogenes,  although  those  diseases  may 
be  complicated  by  genuine  diphtheria.  Streptococci  are  very 
commonly  present  in  the  throat  in  scarlet  fever,1  and  some- 
times occur  in  the  blood.  Some  observers  believe  that  scarlet 
fever  is  caused  by  streptococci.  Streptococci  are  very  often 
found  in  the  pustules  of  small-pox,  and  may  also  appear  in 
the  blood. 

The  streptococcus  pyogenes  is  pathogenic  for  mice  and 
rabbits,  but  the  virulence  is  very  variable.  That  may  some- 
times be  increased  by  passing  through  a  number  of  animals 
in  succession,  but  is  rapidly  lost  in  artificial  cultures.  It  is 
said  that  the  virulence  is  best  maintained  when  cultures  on 
gelatin,  after  forty-eight  hours'  growth,  are  kept  in  a  cool 

'  Weaver,  American  Medicine,  April  18,  1903. 


PATHOGENIC    BACTERIA. 


249 


FIG.  59. 


place,  as  in  the  ice-chest.  Marmorek  undertakes  to  main- 
tain or  increase  the  virulence  by  growing  it  first  in  a  mix- 
ture of  human  blood-serum  (or 
that  of  the  ass  or  the  horse)  with 
bouillon,  and  then  inoculating  it 
into  the  body  of  a  rabbit,  alter- 
nating these  procedures,  to  obtain 
a  culture  of  very  high  virulence. 
A  serum  of  uncertain  value  de- 
rived from  an  immunized  horse 
or  ass  and  intended  to  cure  strep- 
tococcus infection,  has  been  pre- 
pared by  Marmorek. 

A  number  of  other  sera  have 
been  prepared  to  combat  strepto- 
coccus infection.  These  have 
been  used  for  human  cases,  in- 
cluding also  scarlet  fever.  The 
results  appear  somewhat  encour- 
aging, although  still  uncertain. 

It  is  said  that  streptococci  may 
be  agglutinated  by  serum  from 
animals  immunized  to  the  strepto- 
coccus. 

Coley  has  recommended  a 
bouillon  culture  of  streptococcus 
pyogenes  (or  of  erysipelas),  in 
which  the  bacillus  prodigiosus  was 
afterward  grown,  to  be  adminis- 
tered by  injection,  after  steriliza- 


Streptococcus  pyogenes,  cul- 


ture   on    agar     (slightly     en- 

tion  of  the  cultures  by  heat,   in   lar  ed) 
cases   of   inoperable   sarcomatous 

tumors.     These  injections  appear  in  some  cases  to  have  ac- 
complished remarkable  and  wholly  unexplainable  cures. 


250  MANUAL    OF    BACTERIOLOGY. 

Streptococcus  of  Erysipelas. — A  streptococcus  has  been 
derived  from  cases  of  erysipelas  which  in  all  essential  re- 
spects, in  its  morphology,  its  growth  on  culture-media,  its 
behavior  with  stains,  and  its  pathogenic  properties,  is  simi- 
lar to  the  streptococcus  pyogenes.  It  is  probable  that  these 
organisms  are  identical. 

Micrococcus  tetragenus. — Found  in  the  cavities  in  the 
lungs  of  pulmonary  tuberculosis,  in  sputum  and  in  pus. 

FIG.  60. 


Micrococcus  tetragenus  in  pus  from  a  large  abscess  on  the  arm  ;    showing 
capsule,  Gram's  stain  and  eosin.      (X  1000.) 

The  micrococci  are  enclosed  in  a  transparent  capsule,  best 
seen  in  preparations  from  the  tissues  of  inoculated  animals, 
and  are  arranged  in  pairs  or  in  fours ;  about  I  p.  in  diameter ; 
not  motile;  stain  by  Gram's  method.  It  grows  well  at  the 
room  temperature,  but  rather  slowly;  is  facultative  anaero- 
bic; does  not  liquefy  gelatin.  Gelatin  plates  show  little, 
white,  punctiform  colonies,  which,  with  the  low  power,  are 
finely  granular,  and  have  a  peculiar  glassy  shimmer ;  and  in 
stab-cultures  the  growths  appear  as  little  colonies  along 


f' 

PATHOGENIC    BACTERIA.  25! 

the  line  of  puncture.  On  agar,  round  white  colonies  form, 
not  spreading.  It  produces  a  thick,  slimy  film  on  potato  and 
a  broad,  white,  moist  growth  on  blood-serum.  This  organ- 
ism is  only  occasionally  found  in  pus.  It  is  pathogenic  to 
white  mice  and  guinea-pigs,  not  to  gray  mice  and  rabbits. 
It  may  produce  a  septicemia  or  only  a  localized  suppuration 
in  guinea-pigs.  In  white  mice  a  general  septicemia  results, 
when  the  micrococcus  tetragenus  is  found  in  the  blood  and 
in  the  great  viscera.  White  mice  usually  die  in  from  two 
to  six  days;  guinea-pigs  in  from  four  to  eight  days. 

Micrococcus  lanceolatus  (Micrococcus  pneumoniae  crou- 
posse,  Micrococcus  Pasteuri,  Diplococcus  pneumonise,  Micro- 
coccus  of  sputum  septicemia,  Streptococcus  lanceolatus 
Pasteuri,  and  Pneumococcus  of  Frankel). — This  organism 
was  discovered  by  Sternberg  in  his  saliva  in  1880,  and  after- 
ward demonstrated  to  be  the  cause  of  lobar  pneumonia  by 
Frankel  and  Weichselbaum.  The  micrococci  usually  occur  in 
pairs.  The  pair  of  micrococci,  in  its  most  typical  form,  ap- 
pears like  a  couple  of  curved  triangles  with  their  bases  close 
to  each  other.  The  outline  is  usually  described  as  being  lan- 
cet-shaped. The  micrococci  are  frequently  oval  or  round; 
they  often  form  chains.  When  it  is  most  characteristic,  each 
pair  of  micrococci  is  surrounded  with  a  capsule,  which  is 
best  shown  in  preparations  made  from  the  blood  of  infected 
animals  or  from  pneumonic  sputum;  the  capsule  is  not 
usually  seen  in  preparations  made  from  cultures.  For 
methods  of  demonstrating  the  capsule  see  page  57.  The 
pneumococcus  is  not  motile.  It  stains  by  Gram's  method, 
which  also  is  useful  in  demonstrating  the  capsule.  It  is 
facultative  anaerobic.  It  grows  only  at  elevated  tempera- 
tures, preferably  about  35°  to  37°  C.  Gelatin  is  not  lique- 
fied. It  grows  well  upon  agar,  upon  blood-serum  and  upon 
Guarnieri's  medium  (p.  81).  It  does  not  grow  upon  potato. 
Milk  usually  becomes  acid,  and  may  or  may  not  be  coagu- 


252  MANUAL    OF    BACTERIOLOGY. 

lated.  The  colonies  are  seen  in  their  characteristic  form 
upon  agar,  and  are  developed  after  about  forty-eight  hours, 
appearing  as  minute,  whitish,  translucent,  circular  growths. 

It  is  killed  by  an  exposure  to  52°  C.  for  ten  minutes. 

It  is  best  cultivated  from  the  blood  of  an  animal  which 
has  been  infected  with  the  sputum  of  a  case  of  lobar  pneu- 

FIG.  61. 


I 


Pneumococcus  of  Frankel  in  sputum  of  pneumonia,   Gram's  stain  and 
eosin.       (X  1000.) 

monia.  Cultures  need  to  be  transplanted  every  few  days; 
they  cannot  usually  be  propagated  more  than  a  couple  of 
months. 

The  virulence  of  the  organism  for  animals  diminishes 
rapidly  in  cultures.  In  cultures  it  frequently  grows  as  a 
streptococcus.  When  virulent,  it  is  pathogenic  to  mice  and 
rabbits,  less  so  to  guinea-pigs.  In  these  animals  it  is  likely 
to  lead  to  inflammations,  and  to  rapidly  fatal  septicemia 
(twenty-four  to  forty-eight  hours).  The  blood  may  con- 
tain great  numbers  of  the  diplococci.  It  may  be  introduced 


r  , 

PATHOGENIC    BACTERIA.  253 

subcutaneously  or  into  the  peritoneum,  or  by  intravenous 
injection  when  liquid  cultures  are  used.  Its  virulence  is 
very  variable.  In  the  sputum  of  a  case  of  lobar  pneumonia, 
early  in  the  disease,  it  is  likely  to  be  virulent.  The  virulence 
is  best  maintained  by  repeated  inoculations  into  mice  or 

rabbits. 

FIG.  62. 


Pneumococcus    showing    capsule,    from    pleuritic    fluid    of    infected    rabbit, 
stained  by   second  method   of   Hiss.      (X  1000.) 

This  organism  is  detected  very  frequently  in  the  human 
mouth.  When  taken  from  the  mouth  it  is  not,  however, 
pathogenic  to  animals  in  many  instances,  being  found 
virulent  in  only  from  15  to  20  per  cent,  of  human  mouths. 
It  is  the  specific  cause  of  croupous  or  lobar  pneumonia  in 
man.  In  that  disease  the  characteristic  lesion  consists  of  an 
inflammation  of  the  lung,  involving  large  areas,  usually  one 
or  several  lobes.  An  exudate  is  poured  into  the  air-vesicles, 
which  in  the  early  part  of  the  disease  contains  red  blood- 
cells,  imparting  the  rusty  color  to  the  sputum.  The  principal 
element  in  the  exudate  is  fibrin.  The  formation  of  fibrin 
produces  the  liver-like  consolidation  or  "  hepatization." 


254  MANUAL    OF    BACTERIOLOGY. 

The  diplococci  can  readily  be  demonstrated  in  sections  of 
pneumonic  lung,  which  are  best  stained  by  carmine  and 
gentian-violet,  by  the  Gram  method.  Although  the  exudate 
at  first  contains  many  red  blood-cells  and  the  solid  lung 
appears  red,  subsequently  it  becomes  decolorized  and  pre- 
sents a  gray  color.  Many  leucocytes  will  now  be  found  to 
have  migrated  into  the  air-vesicles,  and  the  lung  will  have 
become  relatively  anemic,  instead  of  hyperemic.  Finally, 
the  fibrinous  exudate  and  the  cells  entangled  in  it  become 
softened  and  liquefied.  Some  of  this  liquefied  exudate  is 
absorbed  into  the  lymphatics  in  the  walls  of  the  air-vesicles ; 
part  of  it  is  expectorated. 

The  micrococcus  lanceolatus  can  be  detected  in  large 
numbers,  sometimes  almost  unmixed  with  other  bacteria, 
in  the  rusty  sputum  of  lobar  pneumonia,  often  showing  the 
peculiar  unstained  capsule.  On  account  of  its  liability  to 
be  mixed  with  other  forms  of  bacteria,  its  presence  in  the 
sputum  of  cases  suspected  of  being  pneumonia  is  not  of 
very  great  value  in  differential  diagnosis,  especially  con- 
sidering that  it  is  so  commonly  present  in  the  normal  mouth. 
In  a  suspicious  case  its  appearance  in  sputum  in  nearly  pure 
culture  may  be  significant. 

Cultures  from  the  blood  of  cases  of  pneumonia,  where  a 
large  amount  of  blood  is  taken,  have  shown  the  presence 
of  the  pneumococcus  in  a  considerable  proportion  of  the 
cases,  especially  when  severe  or  fatal. 

The  micrococcus  lanceolatus  is  often  also  the  cause  of 
broncho-pneumonia  and  of  meningitis.  It  produces  inflam- 
mations in  other  situations  as  well,  the  most  important  be- 
ing pleuritis,  pericarditis,  endocarditis  and  arthritis.  The 
micrococcus  lanceolatus  may  produce  pseudomembranous 
inflammation1  and  also  ordinary  suppuration,  although  not 
very  commonly. 

1  Gary  and  Lyon,  American  Journal  Medical  Sciences,  Vol.  122,  1901. 


PATHOGENIC    BACTERIA.  255 

G.  and  F.  Klemperer  claim  to  have  obtained  toxins  from 
cultures  of  the  pneumococcus,  and  to  have  established  im- 
munity in  animals  with  the  development  in  the  blood  of 
antitoxic  substances.  Similar  attempts  have  been  made  by 
Washbourn  and  others,  but  the  interpretation  of  them  at 
the  present  time  is  not  clear.  An  agglutination  reaction 
has  been  described  as  occurring  with  the  pneumococcus,  but 
it  does  not  yet  appear  to  have  any  practical  value  in  diag- 
nosis. 

Organisms  related  to  the  pneumococcus  have  been  de- 
scribed under  the  names  of  pseudopneumococcus1  and  strep- 
tococcus mucosus.2 

The  organism  named  by  Rosenbach,  micrococcus  pyo- 
gcncs  tennis,  is  probably  only  a  variety  of  the  pneumococcus. 

Micrococcus  melitensis. — A  micrococcus  found  by  Bruce 
in  cases  of  Malta  fever.  It  is  a  round  or  slightly  oval 
organism,  about  .5  IJL  in  diameter,  occurring  singly,  in  pairs 
or  in  short  chains.  It  is  usually  said  to  be  non-motile, 
though  flagella  have  been  described.  It  is  stained  by  ordi- 
nary aniline  dyes,  but  not  by  Gram's  method.  It  grows 
slowly,  even  in  the  incubator,  and  more  slowly  at  ordinary 
temperatures.  In  gelatin  the  growth  is  feeble;  there  is  no 
liquefaction.  On  agar  pearly  white  growths  appear  after 
three  or  four  days.  Bouillon  becomes  turbid,  with  a  sedi- 
ment later.  On  potato  there  may  be  slight  invisible  growth. 

Malta  fever  occurs  chiefly  about  the  Mediterranean.  It 
has  been  observed  in  India,  in  the  Philippine  Islands  and  in 
Porto  Rico. 

It  is  a  chronic  febrile  disease,  accompanied  by  pains  in  the 
joints  and  perspiration,  and  not  very  fatal.  At  autopsies 
the  organisms  may  best  be  recovered  from  the  enlarged 
spleen.  Accidental  infection  in  man  has  occurred  from  pure 

1  Richardson,  Journal  Boston  Society  of  Medical  Sciences,  Vol.   V., 
1901. 

2  Howard,  Journal  Medical  Research,  Vol.  VI.,  1901. 


256  MANUAL    OF    BACTERIOLOGY. 

cultures  on  a  number  of  occasions.  The  disease  may  be 
reproduced  in  monkeys  by  inoculation  with  pure  cultures. 
An  agglutination  reaction  occurs  in  this  disease.  The 
diagnosis  is  best  made  by  applying  this  test  to  the  blood- 
serum  of  the  patient,  with  a  known  pure  culture  of  micro- 
coccus  melitensis.1  A  suspension  of  an  agar  culture  is 
made  in  normal  salt  solution.  The  diluted  serum  is  added 
so  as  to  secure  a  dilution  of  about  i  to  50,  but  the  dilutions 
used  have  varied  widely.  Precipitation  quickly  occurs. 
According  to  Craig  the  test  may  be  made  on  a  slide,  examin- 
ing with  the  microscope  as  for  the  typhoid  bacillus  (see 
Serum-test  for  typhoid  fever). 

Diplococcus  intracellularis  meningitidis.2 — Found  in  the 
exudate  of  cerebro-spinal  meningitis  by  Weichselbaum ;  a 
micrococcus  about  the  size  of  the  common  pyogenic  cocci ; 
grows  in  pairs  or  fours,  more  often  in  pairs  consisting  of 
two  hemispheres  separated  by  an  interval  which  does  not 
stain ;  usually  found  within  the  pus-cells,  in  which  respect  it 
resembles  the  gonococcus.  It  is  stained  by  ordinary  methods 
with  the  aniline  dyes,  and  is  decolorized  by  Gram's  method. 
It  does  not  grow  at  the  room  temperature  but  only  in  the 
incubator;  gelatin  is  not  available.  There  is  no  growth  on 
potato  and  scanty  growth  on  agar  or  in  bouillon.  The 
development  is  most  abundant  upon  Loffler's  blood-serum, 
when  round,  white,  shining,  viscid-looking  colonies  with 
sharp  outlines  may  be  seen  in  twenty-four  hours.  The 
serum  is  not  liquefied.  Upon  agar,  or  better  upon  glycerin- 
agar,  the  colonies  are  flat,  round,  translucent,  viscid-look- 

1  Mtisser  and  Sailer,  PhiladelpJiia  Medical  Journal,  December  31,  1898, 
July  8,   1899;  Strong  and  Musgrove,  Ibid.,  November  24,  1900;  Curry, 
Journal  Medical  Research,  Vol.  VI.,  1901. 

2  The  writer  is  indebted  for  the  brief  statement,  which  it  is  possible  to 
give  here,  chiefly  to  the  exhaustive  Report  to  the  Massachusetts  Board 
of  Health  by  Councilman,  Mallory  and  Wright,  1898.     The  photograph 
was  made  from  a  preparation  kindly  furnished  by  Dr.  Mallory. 


PATHOGENIC    BACTERIA.  257 

ing,  under  the  low  power  having  a  yellowish-brown  color. 
The  organism  should  be  transplanted  to  fresh  media  fre- 
quently, as  it  rapidly  loses  its  power  of  reproduction.  Many 
of  the  tubes  inoculated  with  the  original  material  or  with 
pure  cultures  show  no  growth. 

It  is  moderately  pathogenic  for  guinea-pigs  and  rabbits 
when  inoculated  into  the  pleura  or  peritoneum.  Menin- 
gitis and  encephalitis  have  been  produced  in  the  dog  and 
goat  by  inoculation  in  the  meninges. 

FIG.  63. 


Diplococcus  intracellularis  meningitidis  and  pus-cells.      (X  1000.) 

This  organism  appears  to  be  the  principal  if  not  the  only 
cause  of  epidemic  cerebro-spinal  meningitis.  The  lesion 
consists  of  a  purulent  inflammation  of  the  pia  and  arach- 
noid, extending  into  the  brain  substance,  over  the  cord, 
and  along  the  nerves.  General  invasion  of  the  tissues  of 
the  body  seems  not  to  occur,  but  focal  areas  of  pneumonia 
may  be  present.  Spinal  puncture  in  the  lumbar  region  is 
recommended  as  a  means  of  diagnosis.  The  puncture 
should  be  made  early,  and  the  fluid  should  be  examined 
with  the  microscope  and  by  cultures. 


258  MANUAL   OF    BACTERIOLOGY. 

Micrococcus  gonorrheas  (Gonococcus  of  Neisser).— 
Found  in  pus  in  cases  of  gonorrhea.  The  micrococci  gen- 
erally are  in  pairs,  occasionally  in  groups  of  four.  The 
cocci  are  flattened,  the  flattened  sides  facing  each  other,  and 
they  are  often  compared  to  a  pair  of  biscuits.  The  long 
diameter  of  the  pair  of  biscuit-shaped  elements  is  about 
1.25  /*.  The  organisms  are  usually  found  attached  to  the 
epithelial  cells  or  inside  of  the  pus-cells;  they  are  also 
found  in  smaller  numbers  floating  free  in  the  fluid.  They 
stain  with  ordinary  aniline  dyes,  for  example  Loffier's 
methylene-blue,  but  not  by  Gram's  method. 

The  occurrence,  (i)  inside  of  the  pus-cells,  (2)  of  pairs 
of  biscuit-shaped  micrococci  (3)  which  are  not  stained  by 
Gram's  method,  will  serve  to  distinguish  the  gonococcus 
from  all  the  other  ordinary  pus-forming  bacteria.  There 
are  other  diplococci  (pseudo-gonococci),  probably  non- 
pathogenic,  which  have  rarely  been  found  in  the  vulvo- 
vaginal  tract  and  in  the  urethra,  which,  it  is  said,  are  also 
decolorized  by  Gram's  method.  Such  organisms  are  not 
likely  to  present  all  the  points  mentioned  as  characteristic 
of  the  gonococcus.  The  recognition  of  the  gonococcus  in 
the  discharges  of  a  case  of  acute  gonorrhea  is  usually  a 
matter  of  the  greatest  possible  ease.  It  must  be  admitted, 
however,  that  in  cases  having  chronic  discharges,  when  its 
detection  is  most  to  be  desired,  the  diagnosis  may  become 
very  difficult  and  is  frequently  impossible,  except  by  culture- 
methods,  owing  to  secondary  infection  with  the  ordinary 
pus-forming  or  other  bacteria,  which  may  be  present  in 
larger  numbers  than  the  gonococci  themselves. 

The  gonococcus  grows  only  in  the  incubator,  and  cannot 
therefore  be  cultivated  upon  gelatin.  Its  cultivation  is  in 
fact  a  matter  of  some  difficulty.  The  medium  usually 
selected  is  a  mixture  of  agar  with  human  blood-serum.  The 
blood-serum  from  the  placental  blood  or  pleuritic  or  peri- 
toneal transudates,  or  hvdrocele  fluid,  has  been  taken.  The 


PATHOGENIC    BACTERIA. 


259 


addition  of  human  urine,  -sterilized  by  filtration  through 
porcelain,  to  the  mixture  of  blood-serum  and  agar  im- 
proves its  character  according  to  some  writers.  A  con- 
venient medium  is  one  consisting  of  one  part  of  human 
serum  derived  from  a  pleuritic  effusion,  added  to  two  parts 
of  a  2  per  cent,  nutrient  agar.  The  agar  has  previously 

FIG.  64. 


Gonococci    and   pus-cells.      (X  1000.) 

been  sterilized;  the  two  are  mixed  in  tubes  while  fluid; 
they  are  cooled  while  in  an  inclined  position,  and  are 
sterilized  between  65°  and  70°  C.  by  the  fractional  method 
on  six  consecutive  days.  They  are  afterward  tested  in  the 
incubator  for  two  days. 

The  colonies  of  the  gonococcus  are  very  small,  grayish- 
white,  circular,  translucent;  appearing  after  from  twenty- 
four  to  forty-eight  hours.  They  may  attain  a  diameter  of 
i  to  2  mm.  The  gonococcus  will  occasionally  develop  on 
ordinary  glycerin-agar  or  Loffler's  blood-serum  medium, 
but  the  growth  is  likely  to  be  feeble  and  cannot  be  relied 


260  MANUAL    OF    BACTERIOLOGY. 

on.  The  cultures  live  for  a  considerable  time  if  kept  from 
drying.  The  gonococcus  is  not  known  to  produce  ure- 
thritis  or  conjunctivitis  in  any  of  the  lower  animals.  In 
the  peritoneum  it  may  cause  suppurative  inflammation  in 
mice  and  guinea-pigs.  Reproduction  of  the  disease  in 
man  has  been  effected  by  experimental  inoculation  with  pure 
cultures.  Besides  being  the  cause  of  gonorrheal  urethritis 
and  infection  of  the  cervix  uteri,  the  gonococcus  has  been 
isolated  from  cases  of  vaginitis  in  little  girls,  and  from 
gonorrheal  conjunctivitis.  It  has  been  found  to  be  the  cause 
of  many  cases  of  pyosalpinx,  as  well  as  of  gonorrheal  proc- 
titis,  arthritis,  myocarditis  and  endocarditis;  these  condi- 
tions complicating  gonorrhea  may  also  be  secondary  or 
mixed  infections. 

Bacillus  of  Soft  Chancre  (of  Ducrey). — A  small,  oval 
bacillus,  usually  occurring  in  chains.  It  stains  with  ordinary 
aniline  dyes,  but  not  by  Gram's  method.  It  has  been  culti- 
vated on  human  blood-agar  (also  rabbit  blood-agar;  the 
medium  deteriorates  in  a  few  weeks,  Davis).  It  is  culti- 
vated with  difficulty.  It  is  found  in  the  pus  of  soft  chancre 
or  chancroid,  usually  mixed  with  other  organisms.  It  has 
been  demonstrated  in  sections  of  the  ulcers.  There  seems  to 
be  uncertainty  with  respect  to  its  occurrence  in  buboes. 
Ducrey  was  able  to  secure  it  in  pure  culture  by  successive 
inoculations  on  the  human  skin.  Although  this  bacillus  has 
not  yet  been  sufficiently  studied  there  seems  little  doubt  that 
it  is  the  cause  of  soft  chancre.1 

Bacillus  pneumonise  (of  Friedlander) ,  or  Bacillus  nin- 
cosus  capsulatus.2 — A  short  bacillus  with  rounded  ends, 
sometimes  growing  out  to  a  greater  length;  sometimes 
occurring  in  pairs;  surrounded  by  a  capsule  which  is  only 
seen  in  preparations  made  from  the  tissues  of  infected  ani- 

1  Davis,  Journal  Medical  Research,  Vol.  IX.,  1903. 

2  Howard,   Philadelphia  Medical  Journal,  February   19,   1898;   Curry, 
Howard,  Perkins,  Journal  Experimental  Medicine,  Vols.  IV.  and  V. 


PATHOGENIC    BACTERIA.  26l 

mals,  and  is  not  demonstrated  in  cultures.  This  bacillus  is 
not  motile.  It  does  not  form  spores.  It  stains  with  the  ordi- 
nary aniline  dyes,  but  does  not  stain  by  Gram's  method.  It 
is  aerobic  and  facultative  anaerobic.  It  may  be  cultivated 
at  ordinary  temperatures,  but  grows  better  in  the  incubator. 
It  does  not  liquefy  gelatin.  Stick-cultures  in  gelatin  develop 
especially  at  the  point  where  the  puncture  enters  the  surface 
of  the  gelatin,  making  what  is  called  a  "  nail-shaped " 
growth;  the  growth  in  gelatin  is  white;  in  old  cultures  the 
gelatin  acquires  a  brown  color.  It  develops  also  on  the  other 
media.  Dextrose  and  lactose  are  fermented  by  it ;  in  cultures 
on  potato,  gas  is  formed;  milk  is  not  coagulated.  It  does 
not  produce  indol. 

The  thermal  death-point  is  about  56°  C.  It  is  patho- 
genic for  mice,  less  so  for  guinea-pigs  and  rabbits.  This 
bacillus  is  sometimes  found  in  the  healthy  mouth  and  nose. 
It  has  been  known  to  cause  inflammation,  especially  in  the 
vicinity  of  the  mouth,  nose  and  ear,  broncho-pneumonia, 
and  more  rarely  empyema  and  meningitis.  It  was  described 
by  Friedlander  as  the  specific  cause  of  lobar  pneumonia. 
Subsequent  investigations  indicate  that  it  is  comparatively 
seldom  found  in  pneumonia. 

There  are  various  capsulated  bacilli  (capsule  bacilli  of 
R.  Pfeiffer  and  others)  which  closely  resemble  the  bacillus 
of  Friedlander,  and  at  least  belong  to  the  same  group.  The 
bacillus  of  ozaena,  which  has  often  been  found  in  that  dis- 
ease is  very  similar.  B.  lactis  aerogenes  and  B.  coli  com- 
munis  also  have  many  points  in  common  with  the  Fried- 
lander  bacillus. 

Bacillus  of  Rhinoscleroma. — A  short  bacillus  with 
rounded  ends,  often  united  in  pairs,  also  growing  to  a 
greater  length ;  surrounded  by  a  capsule ;  not  motile ;  stained 
by  the  ordinary  aniline  dyes.  It  is  much  like  the  bacillus 
of  Friedlander,  but  some  writers  have  said  that  it  is  not  so 


262  MANUAL    OF    BACTERIOLOGY. 

easily  decolorized  by  Gram's  method;  this  may  be  doubted, 
however.  The  organism  has  been  cultivated.  It  is  faculta- 
tive anaerobic.  It  grows  rapidly,  best  in  the  incubator.  It 
does  not  liquefy  gelatin ;  its  growth  in  gelatin  stick-cultures 
resembles  the  bacillus  of  Friedlander.  It  grows  on  the 
ordinary  media.  Gas  may  be  developed  upon  potato. 

It  is  pathogenic  for  mice  and  guinea-pigs,  less  so  for 
rabbits.  Its  virulence  is  less  than  that  of  Friedlander's 
bacillus. 

It  has  been  obtained  from  the  tissues  of  cases  of  rhino- 
scleroma.  Rhinoscleroma  is  a  disease  characterized  by  a 
chronic  tubercular  thickening  and  swelling  of  the  skin 
around  the  nose  and  similar  swelling  of  the  nasal  mucous 
membrane,  sometimes  followed  by  ulceration.  It  is  com- 
monest in  Austria  and  Italy.  It  has  been  seen  in  America 
only  with  the  greatest  rarity. 

The  organisms  may  be  stained  in  the  diseased  tissues, 
but  their  detection  is  a  matter  of  considerable  difficulty, 
and  they  are  not  always  found.  It  is  not  yet  certain  that 
they  are  the  cause  of  rhinoscleroma. 

Bacillus  pyocyaneus. — A  slim  bacillus  with  rounded 
ends.  It  is  motile.  It  does  not  form  spores.  At  56°  C. 
it  is  killed  in  ten  minutes.  It  is  decolorized  by  Gram's 
method.  It  is  aerobic;  grows  well  at  ordinary  tempera- 
tures; liquefies  gelatin,  and  grows  on  the  ordinary  culture- 
media.  Cultures  present  a  blue  or  green  color,  especially 
in  transparent  media.  This  color  is  not  confined  to  the 
growth  itself,  but  a  blue  or  green  fluorescence  spreads 
over  the  whole  medium.  In  old  agar-cultures  the  color 
may  become  very  dark.  The  pigment  forms  in  the  pres- 
ence of  oxygen,  and  is  due,  at  least  in  part,  to  the  pto- 
maine, pyocyanin.  On  potato  the  growth  is  usually  brown, 
which  may  be  tinged  with  green.  Milk  is  coagulated  and 
peptonized  and  an  acid  reaction  is  developed.  Indol  is 


PATHOGENIC    BACTERIA.  263 

formed    in    Dunham's   peptone   solution.      Blood-serum   is 
liquefied. 

The  bacillus  pyocyaneus  seems  to  be  rather  widely  dis- 
tributed in  nature;  it  has  been  found  on  the  skin,  in  nor- 
mal feces,  also  in  diarrheal  discharges  and  in  dysentery.  It 
is  the  cause  of  the  color  in  blue  or  green  pus.  It  has  fre- 

FIG.  65. 


M'/, 

'         l/^x--- 


%         »  ,   I V      > 

\        *        ' 
C.         *     'I 

*  *        /  I 

..  \*t    ' 


Bacillus  pyocyaneus,  pure  culture.      (X  1000.) 

quently  been  demonstrated  in  pus,  but  oftenest  perhaps,  in 
mixed  infections.  It  has  been  found  in  various  abscesses,  in 
otitis  media,  peritonitis,  appendicitis  and  broncho-pneu- 
monia. It  has  been  known  to  produce  general  septicemia.1 
It  is  pathogenic  for  guinea-pigs  and  rabbits,  in  whom  it  may 
produce  septicemia.  In  animals  it  may  lead  only  to  local 
suppuration,  from  which  they  may  recover,  being  made  im- 

1Lartigau,  Philadelphia  Medical  Journal,  September  17,  1898;  Journal 
Experimental  Medicine,  Vol.  III.,  1898;  Perkins,  Journal  Medical  Re- 
search, Vol.  VI.,  1901. 


264  MANUAL    OF    BACTERIOLOGY. 

mune  to  subsequent  infection  with  this  organism.  It  ap- 
pears that  an  antagonism  exists  between  the  products  of 
the  bacillus  pyocyaneus  and  the  anthrax  bacillus.  Rabbits 
which  have  been  inoculated  with  cultures  of  the  anthrax 
bacillus  may  recover  if  they  are  injected  shortly  after  with 
a  culture  of  the  bacillus  pyocyaneus. 

Bacillus  proteus. — A  bacillus  with  rounded  ends,  vary- 
ing much  in  length,  breadth  .4  to  .6  ^  ;  frequently  appear- 
ing as  short  ovals  like  micrococci;  sometimes  growing  out 
into  long  filaments,  so  that  it  is  said  to  be  pleomorphic. 
Rounded  involution  forms  occur.  It  is  not  stained  by 
Gram's  method.  It  is  motile.  Spore  formation  has  not 
been  observed.  It  is  aerobic  and  facultative  anaerobic.  It 
grows  rapidly  at  ordinary  temperatures.  This  organism 
was  originally  described  by  Hauser  as  three  different 
species — proteus  mdgaris,  which  was  said  to  liquefy  gel- 
atin rapidly,  proteus  mirabilis,  which  liquefied  gelatin 
slowly,  and  proteus  Zenkcri,  which  did  not  liquefy  gelatin. 
It  seems  probable  that  these  organisms  were,  in  fact, 
varieties  of  the  same  species,  now  called  bacillus  proteus. 
Upon  gelatin-plates  the  colonies  present  a  characteristic 
phenomenon,  when  seen  under  the  low  power,  in  the  pro- 
jection of  processes  which  subsequently  change  their  form 
and  position,  and  which  may  become  entirely  detached 
from  the  original  colony,  so  that  the  surface  of  the  gelatin 
may  become  covered  with  so-called  "  swarming  islands." 

The  proteus  grows  on  the  usual  media  tending  to  pro- 
duce foul  odor,  decomposition  and  alkaline  reaction.  In 
urine  it  converts  urea  into  ammonium  carbonate. 

This  organism  is  one  of  those  which  were  formerly  de- 
scribed under  the  name  of  bacterium  termo.  It  is  among 
the  most  common  and  widely-distributed  bacteria.  It  has 
been  found  in  decomposing  animal  and  vegetable  sub- 
stances, in  the  feces,  in  the  urine  in  cystitis,  and  in  the  dis- 


PATHOGENIC    BACTERIA.  265 

charges  of  children  having  cholera  infantum.  It  appears 
that  this  organism  may  occasionally  be  pathogenic  to  man, 
causing  pus-formation,  peritonitis,  and  even  general  infec- 
tion.1 Cultures  injected  in  considerable  amounts  may  be 
pathogenic  to  animals. 

Bacillus  of  Bubonic  Plague. — An  oval  or  short  rod- 
shaped  bacillus,  with  rounded  ends,  sometimes  possessing 
a  capsule.  It  is  not  motile.  It  does  not  form  spores.  With 
the  aniline  dyes  the  ends  stain  more  deeply  than  the  middle, 

FIG.  66. 


X.  %%  *<r 

^*>  »      * 

•    *  ju   i 

Bacillus   of   Bubonic   Plague.     (Yersin.) 

called  polar  staining;  by  Gram's  method  it  is  decolorized. 
It  is  aerobic.  It  grows  at  ordinary  temperatures,  but  better 
in  the  incubator.  It  grows  on  most  media.  The  growths 
are  grayish-white.  Gelatin  and  blood-serum  are  not  lique- 
fied. In  bouillon,  the  medium  remains  clear,  while  a  granu- 
lar deposit  forms  on  the  sides  and  bottom  of  the  tube.  In 
bouillon  to  which  a  few  minute  drops  of  sterile  oil,  as 
cocoanut  oil,  have  been  added,  a  growth  takes  place  from 
the  under  side  of  the  oil  drops.  Such  growths  extend  down, 
and  are  called  stalactite  growths.  The  stalactites  break  off, 
with  the  slightest  disturbance. 
1  Ware,  Annals  of  Surgery,  Vol.  XXXVL,  1902. 


266  MANUAL    OF    BACTERIOLOGY. 

On  agar  containing  3  per  cent,  of  common  salt  remark- 
able involution  forms  appear.  The  stalactite  growths  and 
the  forms  occurring  on  salt-agar  are  considered  the  most 
characteristic  cultural  tests.1 

It  is  sometimes  sensitive  to  drying,  but  may  survive  pro- 
longed drying.  It  is  killed  in  three  to  four  hours  by  direct 
sunlight,  when  spread  in  thin  layers;  in  a  few  minutes  by 
steam  at  100°  C.,  and  in  one  hour  by  I  per  cent,  carbolic 
acid.2  It  is  pathogenic  to  rats,  mice,  guinea-pigs,  rabbits, 
and  a  number  of  other  animals. 

In  man  it  appears  usually  to  enter  through  wounds  of 
the  skin.  Other  possible  avenues  of  infection  are  the  air 
passages,  the  mouth,  and  the  gastrointestinal  tract.  Plague 
is  usually  regarded  as  having  three  different  possible  forms, 
—the  bubonic,  the  pneumonic  and  the  septicemic.  The 
bubonic  form  is  commonest.  The  point  in  the  skin  at  which 
the  inoculation  takes  place  seems  generally  to  exhibit  no 
inflammatory  reaction.  The  lymph-nodes  are  generally 
swollen,  especially  the  deep  inguinal  and  axillary  nodes. 
The  swollen  lymph-nodes  may  suppurate.  The  suppurating 
nodes  often  are  infected  simultaneously  with  micrococci. 
The  bacilli  are  numerous  in  the  enlarged  lymph-nodes,  but 
may  be  detected  in  the  other  organs  of  the  body  and  in  the 
blood.  Fluid  drawn  from  the  buboes  with  a  hypodermic 
needle  may  be  examined  microscopically,  by  cultures  and  by 
inoculation  into  rats  or  guinea-pigs.  In  the  pneumonic  or 
pulmonary  form  the  bacilli  occur  in  the  sputum,  and  may 
be  tested  in  the  same  manner.  This  type  of  the  disease  is 
said  to  be  very  fatal.  In  the  septicemic  form  no  primary 
bubo  is  found,  or  a  bubonic  case  may  become  septicemic. 
This  form  is  very  fatal. 

1  Wilson,  Journal  Medical  Research,  Vol.  VI.,  1901. 

2  See  Viability  of  Bacillus  pestis,  Rosenau,  Marine  Hospital  Service, 
Hygienic  Lab'y,  Bull.  No.  4,  1901. 


PATHOGENIC    BACTERIA.  267 

During  epidemics  of  plague  it  has  been  noted  that  rats 
may  die  in  large  numbers,  and  plague  bacilli  have  often  been 
recovered  from  the  bodies  of  such  rats.  The  systematic 
destruction  by  health  departments  of  all  the  rats  possible  is 
important  where  an  epidemic  is  present  or  is  feared.  The 
same  applies  to  mice.  The  agency  of  fleas  as  carriers  of 
the  bacilli  has  been  suggested,  but  has  not  yet  been  proved ; 
this  is  equally  true  as  to  flies. 

The  greatest  care  must  be  used  in  working  with  the 
bacillus  of  plague.  A  number  of  fatal  results  have  occurred 
through  it  in  laboratory  investigators. 

Haffkine  has  invented  a  method  of  protective  inoculation 
against  plague  by  the  injection  of  cultures  of  plague  bacilli 
which  have  been  sterilized  by  heat,  and  a  little  carbolic  acid 
added.  An  active  immunity,  w-hich  is  quite  lasting,  it  is 
maintained,  may  be  secured  in  some  days.  The  injection 
is  sometimes  followed  by  considerable  constitutional  dis- 
turbance. This  method  seems  likely  to  be  of  considerable 
value. 

Yersin  and  others  have  prepared  protective  sera  on  the 
same  general  principles  used  in  making  other  sera  for  effect- 
ing passive  immunity.  It  is  hoped  they  may  be  useful  in 
producing  quickly  a  temporary  immunity;  and  the  outlook 
for  their  employment  in  the  treatment  of  the  disease  is 
very  encouraging. 

An  agglutination  reaction  has  been  described;  it  is  not 
likely  to  be  cf  great  value  in  diagnosis. 

The  period  of  incubation  in  this  disease  is  from  two  to 
seven  days.  It  has  occasionally  appeared  in  civilized 
countries  during  recent  times  though  not  to  a  very  seri- 
ous extent.  Among  the  localities  of  importance  to  us  it  has 
recently  visited  the  Philippine  Islands,  California  and 
Mexico.  It  has  ravaged  the  southeastern  part  of  Asia 
within  a  few  years.  In  the  Middle  Ages,  and  in  succeeding 
23 


268 


MANUAL    OF    BACTERIOLOGY. 


centuries,  it  devastated  many  of  the  countries  of  Europe, 
where  it  was  one  of  the  most  important  of  the  pestilences 
that  went  in  those  days  by  the  name  of  the  "  Plague."  It 
appears  to  have  been  the  disease  known  in  English  history 
as  the  "  Black  Death."1 

Bacillus  aerogenes  capsulatus. — A  thick  bacillus,  3  to  6 
fj-  in  length,  frequently  capsulated,  discovered  by  Welch  and 
Nuttall.  The  capsules  may  be  found  in  preparations  from 

FIG.  67. 


Bacillus  aerogenes  capsulatus,   Smear-preparation   from   Rabbit's   Liver. 

(X  1000.) 

animal  tissues,  but  rarely  in  cultures.  It  sometimes  forms 
spores  chiefly  in  cultures  on  blood-serum.  The  vegetative 
forms  are  destroyed  at  58°  C.  moist  heat  in  ten  minutes, 
but  the  spores  withstand  boiling  nearly  8  minutes.  It  is  not 
motile.  It  stains  by  Gram's  method.  It  is  anaerobic,  and  is 

1  For  further  details  concerning  plague  consult  articles  by  Barker, 
Novy  and  Flexner,  Trans.  Association  American  Physicians,  1902; 
Calvert,  American  Medicine,  January  24,  1903. 


PATHOGENIC    BACTERIA. 


269 


readily  cultivated  by  Buchner's 
method  for  anaerobes.  It  grows 
best  at  the  body  temperature,  but 
will  grow  at  the  room  tempera- 
ture. It  may  liquefy  gelatin 
slowly  or  not  at  all.  The  growths 
are  whitish.  In  media  containing 
lactose,  dextrose,  or  saccharose  it 
produces  an  abundance  of  gas ;  but 
it  is  also,  according  to  Welch, 
able  to  form  gas  from  proteids. 
Milk  is  coagulated,  and  the  re- 
action becomes  acid.  Gas  forms 
upon  potato,  where  the  growth 
is  thin  and  grayish-white. 

It  occurs  in  the  intestine  of 
man  and  various  other  animals, 
in  soil,  sewage  and  water.  It  is 
not  usually  pathogenic  to  rabbits 
and  mice.  In  guinea-pigs,  spar- 
rows and  pigeons  it  may  produce 
"  gas  phlegmons."  It  has  been 
found  on  numerous  occasions  in 
the  organs  of  human  cadavers 
in  which  a  development  of  gas 
had  taken  place,  producing  bub- 
bles or  cavities  in  the  tissues,  im- 
parting to  them  a  peculiar  spongy 
character  (German,  Schaumor- 
gane).  Probably  this  is  as  a  rule 
a  post-mortem  invasion,  but  there 
is  reason  to  believe  that  in  some 
cases  it  enters  the  circulation  dur- 
ing life.  It  has  been  found  in 


FIG.  68. 


cases  of  emphysematous  gangrene     gas-bubbles. 


Bacillus  aerogenes  capsulatus, 
culture  in  dextrose-agar  showing 


270  MANUAL    OF    BACTERIOLOGY. 

or  cellulitis,  in  various  uterine  infections,  including  physo- 
metra  and  emphysema  of  the  uterine  wall,  in  pneumothorax 
and  pneumoperitonitis,  and  in  other  pathological  conditions 
where  gas  occurs  in  the  tissues.  Exceptionally  it  may  cause 
pus-formation.1  This  bacillus,  or  the  gas  formed  by  it  in 
the  organs  of  human  cadavers,  appears  to  have  furnished 
the  basis  for  some  of  the  cases  in  which  death  has  been 
ascribed  to  the  entrance  of  air  into  the  veins  during  life.  It 
is  the  same  as  the  organism  described  by  E.  Frankel  as 
bacillus  phlegmones  emphysematosae. 

Bacillus  edematis  maligni  (French,  ribrion  scptiquc). 
—A  bacillus  about  I  n  in  breadth,  2  to  10 ,«  in  length,  which 
may  form  threads,  having  rounded  ends  when  occurring 
singly.  It  is  motile,  having  flagella  at  the  sides  and  ends. 
It  forms  spores,  and  may  bulge  at  the  center  in  consequence 
of  the  spores  lorn^ed  there.  It  is  decolorized  by  Gram's 
method.  It  is  a  strict  anaerobe  and  is  best  cultivated  under 
hydrogen.  It  grows  at  ordinary  temperatures,  but  better 
in  the  incubator.  It  liquefies  gelatin  and  blood-serum.  The 
colonies  in  gelatin  are  spherical  and  appear  like  little 
bubbles.  It  grows  well  upon  agar.  Gas  may  be  produced 
in  these  media. 

It  is  found  in  garden  earth,  street  dirt,  and  in  putrefying 
organic  material.  It  is  pathogenic  to  rabbits,  guinea-pigs, 
mice,  pigeons  and  various  other  animals,  including  man. 
Inoculation  results  in  the  production  of  swelling  and  edema, 
spreading  from  the  point  of  inoculation.  Gas  may  be  pro- 
duced in  the  tissue.  It  may  lead  to  widespread  septicemia. 

Bacillus  tetani. — A  slim,  straight  bacillus,  with  rounded 
ends,  which  may  form  in  threads.  It  is  slightly  motile. 
Spores  form  in  culture-media  at  the  end  of  thirty  hours  in 
the  incubator.  The  spores  are  located  at  one  end,  which 
is  swollen,  so  that  in  this  stage  the  organism  has  the  shape 

1  Welch,  Philadelphia  Medical  Journal,  August  4,  1900. 


PATHOGENIC    BACTERIA.  27 1 

of  a  drum-stick.  The  spores  are  extremely  resistant,  and 
in  the  dry  condition  can  exist  for  years.  They  are  killed 
by  moist  heat  at  100°  C.  in  five  minutes;  by  5  per  cent, 
carbolic  acid  in  fifteen  hours ;  by  bichloride  of  mercury, 
i-iooo,  in  three  hours.  The  tetanus  bacillus  stains  by 
Gram's  method.  It  is  a  strict  anaerobe;  it  grows  in  an 
atmosphere  of  hydrogen,  but  not  of  carbon  dioxide.  It  may 
sometimes  be  made  to  grow  very  well  by  Buchner's  method. 

FIG.  69. 

tV 


Tetanus  bacilli,  showing  spores.     (X  1000.) 

It  may  be  cultivated  at  the  room  temperature,  but  better  in 
the  incubator.  It  grows  upon  ordinary  culture-media,  pref- 
erably those  containing  dextrose.  Gelatin  is  liquefied 
slowly;  the  colonies  in  gelatin  present  characteristic  radi- 
ating filaments  and  look  like  a  thistle.  It  grows  on  the 
other  culture-media.  Gas  formation  is  not  pronounced. 

This  organism  appears  to  be  widely  spread  in  external 
nature,  especially  in  the  soil.  It  is  often  found  in  garden 
earth,  and  in  the  feces  of  herbivorous  animals.  McFarland 


272  MANUAL    OF    BACTERIOLOGY. 

believes  that  it  may  occur  in  vaccine  virus  when  that  is  care- 
lessly prepared,  which  would  explain  the  rare  occurrence  of 
tetanus  after  vaccination.1  Tetanus  bacilli  have  been  found 
in  gelatin,  and  it  is  stated  that  the  tetanus  has  followed  the 
injection  of  gelatin  as  a  hemostatic.  The  infection  appears 
almost  always,  if  not  always,  to  be  introduced  through 
some  wound.2  Clinically,  persons  having  the  disease  suffer 
from  spasms  of  the  muscles  about  the  neck  and  the  lower 
jaw  (lock  jaw).  The  spasms  finally  become  general. 

Inoculation  with  a  pure  culture  produces  tetanus  in  mice ; 
also  in  rats,  guinea-pigs  and  rabbits.  The  tetanic  spasms 
begin  in  the  vicinity  of  the  point  of  inoculation  and  after- 
ward become  general.  The  bacilli  are  not  widely  scattered 
through  the  body ;  they  occur  only  in  the  immediate  vicinity 
of  the  original  lesion,  and  there  are  no  important  macro- 
scopic alterations  in  the  internal  viscera. 

Tetanus  is  the  type  of  the  purely  toxic  disease.  Its 
symptoms  may  be  produced  in  animals  by  the  injection  of 
liquid  cultures  which  have  been  deprived  of  their  bacteria 
by  filtration.  The  toxic  substance  appears  not  to  be  a  pto- 
maine, as  was  at  first  supposed,  and  its  exact  nature  is  not 
determined. 

The  poison  is  tremendously  powerful  (see  page  174).  It 
acts  as  an  excitant  to  the  motor  cells  of  the  central  nervous 
system,  especially  the  spinal  cord.  Bolton  and  Fisch  have 
pointed  out  the  possibility  that  horses  used  for  the  prepara- 
tion of  diphtheria  antitoxin  may  be  infected  with  tetanus, 
and  have  tetanus  toxin  in  the  blood. :: 

The  activity  of  the  poison  is  destroyed  by  heat,  and  by 
direct  sunlight ;  various  chemicals  diminish  its  intensity. 

Antitoxins  for  tetanus  have  been  prepared  according  to 
the  principles  employed  for  antitoxins  in  general.  They 

1  Journal  Medical  Research,  Vol.  VII.,  1902. 

2  Wells,  "Fourth  of  July  Tetanus,''  American  Medicine,  June  13,  1903. 

3  Trans.  Association  A^ncrican  Physicians,  1902. 


PATHOGENIC    BACTERIA.  2/3 

have  not  proved  very  markedly  successful.  Unfortunately 
the  disease  is  seldom  suspected  until  a  relatively  large 
amount  of  toxin  has  formed  and  begun  to  manifest  its  action 
in  the  patient's  body.1 

Bacillus  anthracis. — This  is  the  largest  of  the  patho- 
genic bacteria  with  the  exception  of  the  spirillum  of  relaps- 
ing fever,  which  is  longer  but  more  slender.  The  bacillus 
of  anthrax  is  1.25  //  broad,  and  from  3  to  10  n  long.  Ba- 

FIG.   70. 


V 


Anthrax  bacilli,  from  a  pure  culture.2     (X  1000.) 

cillus  aerogenes  capsulatus  is  of  about  the  same  size.  It 
often  forms  long  threads.  A  capsule  is  sometimes  present. 
It  is  not  motile.  It  forms  spores,  which  are  placed  in  the 
centers  of  the  bacilli.  The  spores  form  only  in  the  pres- 
ence of  oxygen;  they  do  not  appear  in  the  body  of  an  in- 
fected animal  during  life.  Anthrax  spores  are  the  most 

1  See  also  Moschkowitz,  Studies  Department  Pathology,  College  Physi- 
cians  and   Surgeons,   New    York,    Vol.    VII.,    1899-1900.      Annals    of 
Surgery,  1900,  p.  442. 

2  The  culture    was   derived    from  a   case   of   malignant   pustule   in   a 
tanner.      The  lesion  was  excised  promptly,  and  the  patient  recovered. 


274  MANUAL    OF    BACTERIOLOGY. 

resistant  of  all  pathogenic  bacteria;  they  have  been  known 
to  withstand  boiling  for  twelve  minutes,1  5  per  cent,  carbolic 
acid  for  forty  days,  and  i-iooo  bichloride  of  mercury  for 
nearly  three  days.  The  anthrax  bacillus  is  aerobic,  although 
not  strictly  so.  It  stains  by  Gram's  method.  It  grows  at 
the  room  temperature,  but  better  in  the  incubator.  It 
liquefies  gelatin  and  blood-serum.  Colonies  in  gelatin  seen 

FIG.  71. 


Anthrax  bacilli,  showing  spores.     (X  1000.) 

under  a  low  power  display  numerous,  irregular,  fine,  hair- 
like  projections;  stab-cultures  in  gelatin  also  present  fine 
projections  passing  from  the  needle-puncture  into  the  solid 
gelatin.  It  grows  on  the  ordinary  culture-media;  the 
growths  are  usually  whitish.  Cultures  on  potato  kept  in 
the  incubator  are  particularly  favorable  to  the  development 
of  spores.  Milk  is  coagulated  and  later  peptonized. 

1  More   than   half   an    hour.      V.    A.    Moore.    "Infections    Disease    of 
Animals,"  1902. 


PATHOGENIC    BACTERIA. 


275 


FlG 


It  is  pathogenic  to  mice  and  guinea-pigs,  less  so  to  rab- 
bits; it  is  also  pathogenic  to  sheep  and  cattle.  Rats  and 
pigeons  are  quite  resistant  but  not  entirely  immune;  cats, 
dogs  and  frogs  are  not  sus- 
ceptible,  or  but  slightly  so. 

Anthrax  is  a  disease 
which  occurs  chiefly  in  cat- 
tle and  sheep.  It  is  com- 
moner on  the  continent  of 
Europe  and  in  Siberia  than 
in  America.  In  susceptible 
animals  inoculated  with 
virulent  cultures  of  the  an- 
thrax bacillus  septicemia  is 
produced.  Large  numbers 

Of   the   bacilli    are    found    in    Colony  of  Anthrax  bacilli  (low  power). 


FIG.   73. 


Bacillus    of   anthrax.       Stick-culture   in    gelatin.       (Giinther.) 

the  blood,  and  may  be  crowded  together  in  the  capillaries 
of  the  liver  and  kidneys.     Men  are  occasionally  affected, 
24 


276  MANUAL   OF   BACTERIOLOGY. 

especially  those  whose  occupations  bring  them  in  contact 
with  cattle  or  with  the  hides  and  wool  of  animals  that  die 
of  the  disease.  The  infection  may  enter  through  wounds 
of  the  skin,  where  it  usually  produces  a  localized  inflam- 
mation known  as  malignant  pustule.  Anthrax  of  the  lungs 
may  be  acquired  by  inhalation  of  material  containing  the 
spores  of  the  bacilli  ("  Wool-sorter's  disease  ").  Infection 
FlG  by  way  of  the  intestine  oc- 

curs occasionally  but  is  less 
common.  Laboratory 
workers  engaged  in  study- 
ing the  anthrax  bacillus 
have  been  accidentally  in- 
fected in  a  number  of  in- 
stances. 

The  anthrax  bacillus, 
owing  to  its  large  size, 
was  the  first  of  the  patho- 
genic bacteria  to  be  recog- 
nized, and  its  study  has 

Anthrax      bacilli      with      square      or     furnished       the      basis       for 
slightly  concave  ends   sometimes  seen  ;  ,  r 

much    of    our    knowledge 

fuchsin  stain.      (X  1000.)  .  & 

concerning    the    infectious 

diseases.  It  was  for  anthrax  that  Pasteur  developed  the 
idea  of  making  a  protective  vaccine,  shortly  after  he  had 
invented  a  similar  vaccine  for  chicken-cholera.  There  is 
some  danger  attending  its  use. 

In  order  to  obtain  material  free  from  spores  the  blood  of 
an  animal  which  has  recently  died  of  anthrax  is  taken, 
because  anthrax  spores  do  not  form  in  the  living  body. 
Cultures  made  in  bouillon  are  kept  at  a  temperature  of 
from  42°  to  43°  C.  At  this  temperature  spores  do  not 
form,  while  the  virulence  of  the  anthrax  bacillus  becomes 
gradually  diminished.  In  time  the  virulence  is  so  far 
diminished  that  rabbits  will  survive  inoculation,  and  even- 


PATHOGENIC    BACTERIA.  277 

tually  also  mice  and  guinea-pigs,  which  are  extremely  sus- 
ceptible to  anthrax.  Small  doses  of  a  culture  of  extremely 
weak  virulence  are  given  to  the  animals  which  it  is  desired 
to  protect,  like  cattle  and  sheep  (never  human  beings),  and 
subsequently  a  somewhat  more  virulent  culture  is  employed.1 

FI<;.  75 


Anthrax   bacilli   in  the  capillaries   of  the   liver  of  a  mouse,   sketched  from 
a  section  stained  with   fuchdn. 

Bacillus  influenzas. — A  small  bacillus,  .2  to  .3  j*.  by  .5  IJL 
with  rounded  ends.  It  does  not  form  spores,  is  not  motile, 
and  is  decolorized  by  Gram's  method.  It  is  aerobic,  grows 
only  in  the  incubator,  and  upon  media  containing  hemo- 
globin. The  medium  is  prepared  by  smearing  sterile  blood 
over  the  surface  of  a  tube  of  agar.  Fresh,  uncoagulated 
blood  may,  with  care,  be  mixed  with  melted  agar  sufficiently 
cooled;  the  mixture  may  be  poured  into  tubes  and  slanted; 

1  For  details  as  to  the  results  of  this  method,  see  V.  A.  Moore,  "  In- 
fectious Diseases  of  Animals,"  1902.  For  other  and  unique  researches  on 
immunity  for  anthrax  see  Emmerich,  Centralblatt  f.  Bakteriologie,  Orig. 
Bd.  XXXII,  p.  821. 


2/8  MANUAL    OF    BACTERIOLOGY. 

the  tubes  should  be  tested  in  the  incubator  before  using. 
The  blood  of  some  animals,  as  the  pigeon  and  rabbit,  may 
be  used  instead  or  human  blood.1  The  colonies  are  small 
and  transparent,  looking  like  little  drops  of  water,  not  be- 
coming confluent. 

Of  a  large  number  of  bacilli,  the  majority  are  destroyed 
in  twenty-four  hours  or  less  by  drying.  They  die  out  in 
a  similar  manner  in  water.  Experiments  upon  animals 
appear  up  to  this  time  not  to  have  been  very  convincing. 
In  diagnosis,  the  sputum  should  be  carefully  collected  in  a 
sterile  bottle.  If  the  particles  of  sputum  are  likely  to  have 
become  contaminated,  rinse  in  sterile  water.  Inoculate  on 
agar  and  on  blood-agar.  The  influenza  bacillus  should  grow 
only  on  blood-agar  and  have  the  other  characters  above 
mentioned.  As  far  as  is  known,  this  organism  grows  only 
in  man,  and  not  outside  of  the  human  body.  In  cases  of 
influenza  it  is  found  in  the  mucous  discharges,  and  in  the 
bronchi  and  lungs.  It  is  the  predominating  organism  in 
some  cases  of  bronchitis.2  According  to  Canon,  the  bacilli 
may  sometimes  be  found  in  the  blood. 

Bacillus  diphtherias  (Klebs-Loffler). — A  straight  or 
slightly-curved  bacillus,  usually  1.2  to  2.5  ft  in  length,  with 
rounded  or  slightly  pointed  ends,  remarkable  for  showing- 
irregularities  of  form,  sometimes  being  club-shaped  or 
spindle-shaped;  branching  forms  have  been  found.3  It  is 
not  motile,  and  does  not  form  spores.  It  retains  its  color 
after  Gram's  method,  but  it  is  best  stained  with  watery 
solutions  of  the  aniline  dyes,  especially  LofHer's  alkaline 
methylene-blue.  Very  characteristic  pictures  are  obtained 
by  the  method  of  Neisser : 

». 

1  Ccntralblatt  f.  Baktcriologic,  Bel.  XXXII.,  Orig.  p.  667. 

2  See  Lord,  Hoston  Medical  and  Surgical  Journal,  December  8,   1902. 
;:  Hill,  Journal  Medical  Research,  Vol.  VII,  1902. 


PATHOGENIC    BACTERIA.  279! 

SOLUTION  No.  i. 

Methylene-blue    I 

Alcohol   (96  per  cent. ) 20 

Distilled  water  95° 

Glacial  acetic  acid 50 

SOLUTION  No.  2. 

Bismarck  brown I 

Boiling  distilled  water 500 

Stain  the  cover-glass  preparation  which  has  been  fixed 
in  the  flame  in  No.  i  one  to  three  seconds ;  wash  in  water ; 
stain  in  No.  2  three  to  five  seconds;  wash  in  water;  mount 

FIG.  76. 


*  »         -*j  -* 

[  r     O, '  \  v. 


Bacillus   of  diphtheria.      (X  1000.) 

as  usual.     The  body  of  the  bacillus  is  stained  pale  brown, 
with  dark  blue  spots,  especially  at  the  ends.      (Fig.  77.) 

The  diphtheria  bacillus  is  peculiar  in  staining  irregularly ; 
certain  spots  stain  more  sharply  than  other  portions,  and 
darkly-stained  spots  are  likely  to  occur  at  the  ends.  It  is 
facultative  anaerobic.  It  grows  most  rapidly  in  the  incuba- 
tor, and  slowly,  or  not  at  all,  below  20°  C.  Gelatin  is  not 
liquefied.  It  may  be  cultivated  on  various  alkaline  culture- 


28O  MANUAL    OF    BACTERIOLOGY. 

media,  but  grows  best  on  Loffler's  blood-serum  mixture. 
On  this  medium  the  growth  consists  of  small  white  or  cream- 
colored,  slightly  elevated  colonies,  which  may  become  con- 
fluent. The  morphology  of  the  bacillus  is  most  character- 


Bacillus  of  diphtheria  stained  by  Xeisser's  method.     (X  1000.) 

istic  when  it  is  cultivated  on  blood-serum.  It  also  grows 
upon  glycerine-agar.  On  potato  it  produces  an  invisible 
growth  (see  Bacillus  of  Typhoid  Fever).  In  alkaline 
bouillon  containing  dextrose  (or  muscle-sugar)  the  reaction 
becomes  acid  in  forty-eight  hours.  The  reaction  of  the 
bouillon  subsequently  becomes  alkaline.  The  growth  may 
form  a  pellicle  over  the  surface  of  the  bouillon.  It  has  also 
been  successfully  cultivated  on  various  media  to  which  egg- 
albumen  has  been  added. 

It  is  killed  by  a  temperature  of  58°  C.  in  ten  minutes. 
It  resists  desiccation  well. 

Bacteriological  diagnosis  of  Diphtheria. — In  many  large 
cities  the  bacteriological  diagnosis  of  diphtheria  is  under- 
taken bv  boards  of  health.  The  methods  used  differ  some- 


PATHOGENIC    BACTERIA. 


28l 


FIG.  78. 


what  in  detail,  but  are  similar  in  the  main,  and  are  based 
upon  the  procedure  devised  by  Biggs  and  Park  for  the 
Board  of  Health  of  New  York  City.  Two  tubes  are  fur- 
nished in  a  box.  The  tubes  are  like  ordinary  test-tubes, 
about  three  inches  in  length,  rather 
heavy,  and  without  a  flange.  Both  are 
plugged  with  cotton.  One  contains  slant- 
ed and  sterilized  Loffler's  blood-serum 
mixture;  the  other  contains  a  steel  rod, 
around  the  lower  end  of  which  a  pledget 
of  absorbent  cotton  has  been  wound  and 
the  tube  afterward  sterilized.  The  swab 
is  wiped  over  the  suspected  region  in 
the  throat,  taking  care  that  it  touches 
nothing  else,  and  is  then  rubbed  over  the 
surface  of  the  blood-serum  mixture.  The 
swab  is  returned  to  its  test-tube  and  the 
cotton  plugs  are  returned  to  their  respec- 
tive tubes.  The  plugs,  of  course,  are  held 
in  the  fingers  during  the  operation,  and 
care  must  be  taken  that  the  portion  of  the 
plug  thatgoes  into  the  tube  touches  neither  tube  usf;.n  ,th.c 

nosis  of  diphtheria. 

the  finger  nor  any  other  object.  The  prin- 
ciples, in  fact,  are  the  same  as  those  laid  down  in  general  for 
the  inoculation  of  culture-tubes  with  bacteria  (see  page  84). 
In  board  of  health  work  these  tubes  are  returned  to  the 
office.  When  it  is  desirable,  a  second  tube  may  be  inoculated 
from  the  swab.  The  tubes  are  placed  in  the  incubator, 
where  they  remain  for  from  twelve  to  twenty-four  hours, 
and  a  microscopical  examination  is  then  made  of  smear  pre- 
parations stained  with  Loffler's  methylene-blue.  On  Loffler's 
blood-serum  kept  in  the  incubator  the  bacillus  of  diphtheria 
grows  more  rapidly  than  the  other  organisms  which  are  ordi- 
narily encountered  in  the  throat,  a  property  which  to  a  cer- 
tain extent  sifts  it  out,  as  it  were,  from  them,  and  makes  its 


Swab    and    culture- 


282 


MANUAL    OF    BACTERIOLOGY. 


recognition  with  the  microscope  easy  in  most  cases.     The 
growth,  furthermore,  is  quite  characteristic,  and  its  nature 

can  be  predicted  with  consider- 
able accuracy,  even  without  mi- 
croscopical examination,  by  one 
who  has  had  much  practice. 
Colonies  of  streptococci  fre- 
quently look  very  like  those  of 
the  bacillus  of  diphtheria,  but 
these  two  are  easily  distin- 
guished from  each  other  with 
the  microscope.  The  diagnosis 
of  the  diphtheria  bacillus,  then, 
is  made  from  the  character  of 
the  growth  upon  blood-serum 
and  the  microscopical  exami- 
nation, taking  into  account  the 
size  and  shape  of  the  bacilli, 
with  the  frequent  occurrence 
of  irregular  forms  and  the  pe- 
culiar irregularities  in  stain- 
ing. In  doubtful  cases  a  sec- 
ond culture  should  be  made 
from  the  throat. 

The   very   large   number   of 
examinations    that    have    been 
made    by    various    boards    of 
health,  have  shown  that  pseudo- 
membranous   inflammations  of 
the  throat  are  sometimes  caused 
by    streptococci    alone,    or    by 
They  have  also  shown  that  the 
diphtheria   bacillus   may  persist    in   the   throat   for  a   long 

1  Bissell.  .Ifedictil  NeiVS,  May  31,  1902;  American  Journal  Medical 
Sciences,  February,  1903,  Review  of  Work  of  Massachusetts  Boards  of 
Health. 


Bacillus   of  diphtheria,   culture 
on  glycerine-agar. 

other  pyogenic  bacteria.1 


.PATHOGENIC    BACTERIA. 

time,  occasionally  several  weeks  after  the  patient  has 
apparently  recovered;  also  that  diphtheria  bacilli  are 
occasionally  found  in  the  throat  when  there  is  an  in- 
flammatory condition  without  any  pseudo-membrane, 
and  that  they  sometimes  appear  in  an  apparently  healthy 
throat,  especially  in  children  who  have  been  associated  with 
cases  of  diphtheria.  It  has  been  found  that  bacilli  some- 
times occur  in  the  throat  which  have  all  the  morphological 
and  cultural  properties  of  the  diphtheria  bacillus,  but  which 
are  devoid  of  virulence  when  tested  upon  animals,  i.  e.t 
do  not  produce  diphtheria  toxin.  Such  diphtheria  bacilli 
have  frequently  been  called  pseudo-diphtheria  bacilli.  A 
bacillus  closely  resembling  the  diphtheria  bacillus,  but  with- 
out virulence,  has  been  found  in  xerosis  of  the  conjunctiva. 
It  is  called  the  xcrosis  bacillus.  If  not  a  transformed  diph- 
theria bacillus,  it  is  at  least  closely  related.  The  diphtheria 
bacillus  is  subject  to  wide  variations  in  morphology,  so  that, 
in  dealing  with  unknown  cultures  where  the  forms  of  the 
bacilli  are  not  characteristic  and  injection  into  animals  is 
without  result,  it  may  be  difficult  to  decide  whether  or  not 
the  organisms  are  diphtheria  bacilli.  Consequently  another 
view  with  regard  to  pseudo-diphtheria  bacilli  has  arisen. 
While  recognizing  that  avirulent  diphtheria  bacilli  occur, 
it  is  also  claimed  that  a  distinct  pseudo-diphtheria  bacillus 
exists,  different  from  the  diphtheria  bacillus,  though  re- 
sembling it.  It  is  shorter,  stains  more  evenly,  shows  no 
polar  granules  by  Neissers  method  of  staining,  does  not 
produce  acid  in  dextrose-bouillon,  and  is  not  pathogenic  to 
animals.  It  is  found  occasionally  in  the  nose  and  throat, 
and  has  no  connection  with  diphtheria,  according  to  this 
view.1 

1The  different  sides  of  this  question  will  be  found  fully  discussed 
by  the  following:  Wesbrook,  Wilson  and  McDaniel,  Trans.  Association. 
American  Physicians,  1900;  Gorham,  Journal  Medical  Research,  Vol. 
VI.,  1900;  A.  Williams,  Ibid.,  Vol.  VIII.,  1902;  Denny,  Ibid.,  Vol.  IX., 
1903. 

25 


284  MANUAL   OF    BACTERIOLOGY. 

The  diphtheria  bacillus  is  pathogenic  to  animals.  When 
it  is  injected  into  them  it  produces  a  toxemia.  In  the 
guinea-pig,  which  is  especially  susceptible,  local  inflamma- 
tion results,  and  death  occurs  usually  in  two  or  three  days. 
The  bacilli  are  found  to  be  confined  to  the  vicinity  of  the 
wound,  and  not  usually  to  be  disseminated  throughout  the 
whole  body.  The  death  of  the  animal,  therefore,  is  due  to 
the  poisons  elaborated  by  the  diphtheria  bacilli — either 
poisons  introduced  at  the  original  injection,  or  substances 
produced  by  the  bacilli  which  may  have  multiplied  in  the 
animal's  body.  The  internal  viscera,  especially  the  liver, 
often  exhibit  small  areas  consisting  of  necrotic  cells;  a 
transudation  of  serum  takes  place  in  the  great  serous  cavi- 
ties, and  the  lymph-nodes  are  swollen.  A  genuine  diph- 
theritic membrane  may  be  produced  on  the  trachea  of  a 
young  kitten  by  rubbing  into  it  a  part  of  a  culture  of  the 
diphtheria  bacillus. 

As  is  well  known,  the  pseudo-membranous  affection  pro- 
duced by  the  diphtheria  bacillus  in  man  is  generally  seen  in 
the  larynx  and  pharynx.  Membranous  rhinitis  is  also 
caused  by  the  diphtheria  bacillus.  On  the  other  hand, 
pseudo-membranous  affections  of  the  larynx  and  pharynx 
may  be  produced  by  streptococci.  Pseudo-membranes 
occurring  in  the  throat  during  scarlet  fever  and  measles 
may  be  due  to  the  diphtheria  bacillus,  but  are  more 
often  caused  by  streptococci.  The  affection  known  as  mem- 
branous croup  is  usually  diphtheria  of  the  larynx,  produced 
by  the  diphtheria  bacillus.  The  diphtheria  bacillus  is  a  rare 
cause  of  puerperal  fever.  Although  the  uninjured  skin  is 
not  attacked  by  the  diphtheria  bacillus,  it  may  be  present  in 
pseudo-membranes  on  wounded  surfaces,  usually  in  con- 
nection with  diphtheria  in  the  throat.  Most  pseudo-mem- 
branes formed  upon  wounds  of  the  skin  are  produced  by 
other  bacteria  than  the  diphtheria  bacillus,  as  is  also  the  case 


PATHOGENIC    BACTERIA.  285 

with  the  pseudo-membranous  inflammations  of  the  intestines 
and  bladder.  Although  such  inflammations  are  often  called 
"  diphtheritic,"  it  must  be  remembered  that  the  expression 
is  used  in  an  anatomical  sense,  meaning  that  a  fibrinous 
pseudo-membrane  has  formed,  extending  deeply  into  the 
tissues,  which  is  not  necessarily  caused  by  the  diphtheria 
bacillus. 

In  cases  of  diphtheria  in  man,1  the  diphtheria  bacillus  is 
generally  found  limited  to  the  vicinity  of  the  pseudo-mem- 
brane, and  at  autopsies  it  is  not  usually  found  in  the  in- 
ternal viscera,  excepting  in  the  lungs,  where  diphtheria 
bacilli  may  or  may  not  be  present  when  diphtheria  is  com- 
plicated with  broncho-pneumonia.  The  general  symptoms 
of  the  disease,  including  the  paralysis  which  sometimes 
follows  it,  are  due  to  the  toxins  produced  by  the  bacilli  in 
the  throat. 

Diphtheria  antitoxin.  It  is  necessary  first  to  obtain  the  toxin  pro- 
duced by  diphtheria  bacilli  in  a  concentrated  form.  Virulent  diphtheria 
bacilli  are  cultivated  in  alkaline  bouillon,  in  flasks  plugged  with  cotton, 
exposing  a  large  surface  to  the  air.  The  cultures  are  grown  in  the 
incubator.  After  five  to  ten  days  they  are  ready,  and  are  filtered  through 
porcelain.  The  filtrate  contains  the  toxin.  The  animal  usually  em- 
ployed is  the  horse,  which  should  be  healthy;  the  presence  of  tubercu- 
losis and  glanders  should  have  been  excluded,  testing  with  tuberculin 
and  mallein ;  tetanus  should  also  be  considered,  see  page  272.  The  toxin 
is  injected  into  the  horse  in  small  doses — about  i  c.c.  of  the  filtrate  from 
the  bouillon  culture.  The  dose  depends  on  the  strength  of  the  toxin. 

The  injection  is  repeated  at  intervals  of  about  one  week,  using  larger 
and  larger  doses,  until  the  animal  is  able  to  tolerate  a  very  large  dose 
indeed — as  much  as  300  c.c.,  or  even  more.  If  the  treatment  is  suc- 
cessful the  general  condition  of  the  animal  should  not  suffer.  The 

1  For  a  full  study  of  the  lesions  of  diphtheria  see  the  Monograph  of 
Councilman,  Mallory  and  Pearce,  Boston,  1901. 

2  See  articles  by  Park,  A.  Williams,  Atkinson  and  T.  Smith,  Journal  of 
Experimental  Medicine,  Vol.  I.,  p.  164;  Vol.  III.,  p.  513;  Vol.  IV.,  pp. 
373  and  649;  Journal  Medical  Research,  Vol.  IX.,  p.  173. 

3  W.  H.  Park  adds  10  per  cent,  of  a  5  per  cent,  solution  of  carbolic  acid 
to  kill  the  bacilli,  and  filters  through  paper  on  the  following  day;    after 
adding  carbolic  acid  the  Berkenfeld  filter  may  be  used  with  advantage 
instead  of  filter-paper. 


286  MANUAL    OF    BACTERIOLOGY. 

injections  last  over  a  long  period — usually  about  two  or  three  months. 
The  general  condition  of  the  animal  remaining  good,  the  toleration  of 
these  large  doses  of  toxin  is  presumed  to  indicate  the  existence  of  a  con- 
centrated antitoxic  substance  in  the  blood.  Small  quantities  of  blood 
may  be  withdrawn  from  time  to  time,  and  the  serum  tested  for  its 
antitoxic  strength.  When  a  satisfactory  serum  has  been  attained,  the 
animal  may  be  bled  and  the  serum  saved  for  therapeutic  purposes. 
Through  an  incision  in  the  skin  a  trochar  is  inserted  into  the  jugular 
vein.  The  blood  is  conducted  into  sterilized  flasks  with  every  precau- 
tion to  insure  sterility.  The  blood  is  allowed  to  coagulate  and  is  placed 
for  a  time  in  the  ice-chest.  The  serum  is  then  withdrawn  with  steril- 
ized pipettes.  Small  amounts  of  chemical  germicides,  as  carbolic  acid 
or  chloroform,  are  sometimes  added  to  assist  in  preserving  it.  This 
serum  is  the  so-called  antitoxin  used  in  medical  practice. 

A  standard  to  express  the  potency  of  the  serum,  called  an  immunity 
unit,  has  been  devised  by  Behring  and  modified  by  Ehrlich.  Such  an 
immunity  or  antitoxic  unit  is  the  amount  of  antitoxic  principle  con- 
tained in  that  quantity  of  serum,  which,  when  mixed  and  injected  with 
one  hundred  times  the  fatal  dose  of  toxin  will  preserve  the  life  of  a 
guinea-pig  weighing  250  grains  for  four  days. 

It  has  been  proposed  to  modify  this  test  so  as  to  lessen  certain 
sources  of  error.  According  to  the  new  method,  one  hundred  fatal 
doses  of  toxin  would  not  be  used,  but  the  amount  of  toxin,  which, 
when  mixed  with  one  unit  of  some  special  and  previously  standardized1 
antitoxin  and  injected  into  a  guinea-pig  weighing  250  grams,  will  kill 
the  animal  in  four  days.  This  procedure  would  partly  do  away  with 
inaccuracies,  unavoidable  where  one  hundred  fatal  doses  of  toxin  are 
used  as  a  standard,  resulting  from  changes  that  occur  in  the  toxin 
(development  of  toxoids,  presence  of  IOXOIK-S,  etc,  Ehrlich). 

It  has  been  found  possible  to  prepare  antitoxin  of  a  high 
degree  of  concentration,  so  that  500  to  1,500  units  may  be 
contained  in  a  quantity  of  serum  which  it  is  practicable  to 
give  at  a  single  hypodermic  injection.  The  large  volume  of 
statistics  that  have  been  collected  from  hospitals,  and  from 
physicians  in  private  practice  indicates  that  the  use  of  this 
serum  has  effected  a  very  great  reduction  in  the  mortality 
from  diphtheria. 

'The  standard  largely  used  in  this  country  is  an  antitoxin  prepared 
for  this  purpose  by  the  lustitut  fiir  cxpcrimcntcllc  Tlicrapic,  Frankfort 
a  Main,  (iermany.  Prof.  Ehrlich,  Director. 


f 

PATHOGENIC    BACTERIA.  287 

Bacillus  tuberculosis.  —  A  slim  bacillus  1.5  to  4  ^  in 
length,  which  very  frequently  presents  a  beaded  appear- 
ance, owing  to  its  being  dotted  with  bright,  shining  spots. 
Branching  forms  have  been  described.  The  tubercle  bacillus 
is  considered  by  some  to  be  a  member  of  the  actinomyces 
group.  It  is  not  motile.  It  has  not  been  proved  that  spores 
are  formed;  nevertheless  certain  structures,  like  caseous 
lymph-nodes,  have  been  shown  to  be  capable  of  infecting 
guinea-pigs  with  tuberculosis,  although  tubercle  bacilli  could 


FIG.  80. 


' 


Bacillus  tuberculosis,   from  a  pure  culture.       (X  1000.) 

not  be  demonstrated  in  them  with  the  microscope.  This 
makes  it  seem  possible  that  the  organisms  were  present  as 
spores  which  eluded  the  microscopical  examination.  The 
tubercle  bacilli  stain  with  the  ordinary  aniline  clyes  and  by 
Gram's  method.  As  has  already  been  stated,  when  stained 
with  aniline-water  dyes  or  carbol-fuchsin  they  are  not 
readily  decolorized  by  acids  and  alcohol,  which  fact  distin- 
guishes them  from  all  other  known  bacteria  excepting  the 
leprosy  bacillus,  the  bacillus  of  smegma,  possibly  the  bacillus 


r 

.  \A  x 

\f 


288  MANUAL    OF    BACTERIOLOGY. 

of  syphilis  (Lustgarten),  and  certain  bacilli  found  in  milk, 
butter  and  cow-dung  and  on  various  grasses.  All  of  these 
may  resist  decolorization  by  acids  or  alcohol,  and  some 
resist  both.  They  must  always  be  kept  in  mind  in  making 
a  diagnosis  of  tuberculosis.  (See  pages  44  and  295.)  In 
FIG.  8r.  examining  sputum  it  is  particularly 

important  to  bear  in  mind  that  acid- 
proof  bacilli,  resembling  tubercle 
.  bacilli,  have  rarely  been  found  in 

r  cases    of    gangrene    of    the    lung. 

They  are  likely  to  be  longer  than 
>         tubercle    bacilli,    and    branch    more 
/  often,    besides    being   less    resistant 

to     decolorization.1     The     tubercle 
Branching     form     of        bacilli  appear  to  owe  their  peculiar 

tubercle   bacillus    from   a  .     .  .  f    , 

staining    properties    to    fatty    sub- 
culture.    (X  1000.)  l  J 

stances  contained  in  the  bodies  of  the 

bacilli.  In  stained  preparations  the  bacillus  usually  appears 
very  distinctly  beaded,  owing  to  the  presence  of  stained  areas 
which  alternate  with  unstained  areas;  these  unstained  areas 
have  been  considered  by  some  to  be  spores. 

The  bacillus  tuberculosis  is  aerobic.  It  is  cultivated  with 
considerable  difficulty,  best  at  about  38°  C.  It  does  not 
grow  at  a  temperature  below  29°  C.,  and  cannot  therefore 
be  cultivated  upon  gelatin.  It  grows  best  upon  blood-serum, 
where  the  growth  becomes  visible  in  from  ten  to  fourteen 
days  in  the  incubator.  It  forms  a  dry,  mealy,  scaly  mass, 
elevated  above  the  surface,  of  a  grayish-brown  color.  It  also 
grows  upon  glycerin-agar  ;  or  glycerin-bouillon,  on  which 
it  forms  a  pellicle;  upon  potato;  upon  milk  containing  i  per 
cent,  of  agar  and  upon  coagulated  egg  (see  page  81).  It 
is  important  to  have  the  medium  moist.  It  can  be  cultivated 

1  Ophiils,   Journal   Medical   Research,    Vol.    VIII.,    1902;    Ohlmacher, 
Journal  ^linerican  Medical  Association,   1901. 


f 

PATHOGENIC    BACTERIA.  280 

from  tuberculous  sputum  only  with  very  great  difficulty.  It 
is  best  to  obtain  it  from  the  tissues  of  an  animal  that  has 
died  of  tuberculosis,  where  the  tubercle  bacilli  may  be  found 
unmixed  with  other  bacteria.  Pieces  of  tissue  should  be 
taken  with  the  precautions  necessary  to  avoid  contamina- 
tion, and  should  be  broken  up  and  rubbed  over  the  surface 
of  the  medium.  The  tubes  must  be  closed  with  sealing-wax, 
paraffin  or  rubber  stoppers,  or  covered  with  rubber  caps, 


FIG.  82. 


Bacillus  tuberculosis   in   sputum,  stained   with    fuchsin   and   methylene  blue. 
Photomicrograph   in  two   colors.      (X  1000.) 

to  prevent  drying  in  the  incubator.  If  rubber  caps  are  used 
they  should  first  be  left  in  i-iooo  bichloride  of  mercury  for 
an  hour,  and  the  cotton  plug  should  be  burned  before  putting 
on  the  rubber  cap.  A  number  of  tubes  should  be  inoculated, 
'using  rather  large  particles  of  the  tuberculous  material. 
Among  the  tubes  inoculated,  many  will  fail  to  present  any 
growth.  After  the  organism  has  once  been  grown  upon 
a  culture-medium  it  may  be  propagated  with  less  difficulty. 
It  is  best  cultivated  the  first  time  upon  blood-serum. 


290  MANUAL   OF    BACTERIOLOGY. 

It  is  killed  by  5  per  cent,  solution  of  carbolic  acid  in  a 
few  minutes.  In  sputum  it  is  destroyed  in  twenty-four 
hours  by  a  three  per  cent,  solution  of  carbolic  acid.  It 
resists  desiccation  for  months,  but  is  killed  in  some  hours 
by  direct  sunlight.  It  is  destroyed  in  a  few  minutes  by 
boiling.. 

It  is  not  known  to  grow  outside  of  the  animal  body.  It 
is  the  cause  of  tuberculosis  in  man.  It  produces  tubercu- 
losis in  apes,  cows,  sheep,  horses,  rabbits,  guinea-pigs,  cats, 
field-mice,  and  occasionally  in  other  animals.  Guinea- 
pigs  and  rabbits  are  extremely  susceptible.  A  guinea-pig  in- 
oculated with  tuberculous  sputum  (provided  it  does  not  die 
of  septicemia,  due  to  the  pyogenic  micrococci  which  are  fre- 
quently present  in  sputum)  will  present  a  swelling  of  the 
neighboring  lymph-nodes  in  the  course  of  two  to  four  weeks, 
and  will  die  as  a  rule  in  from  four  to  eight  weeks,  although 
the  time  may  be  longer. 

Tuberculosis  in  cattle  ((lerman,  J'crJsuc/if}  is  characterized  by  large. 
nodular  lesions,  \\itb  a  marked  tendency  to  become  fibrous,  caseous 
and  calcified.  Tbe  tubercle  bacilli  of  cattle  differ  somewhat  from  those 
of  human  tuberculosis,  as  was  noted  by  T.  Smith.1  \Yhether  or  not 
men  could  be  infected  with  bovine  tubercle  bacilli,  has  been  a  question 
that  has  been  warmly  debated  in  recent  years.  It  seems  to  have  been 
shown  that  such  infection  is  possible:  also  that  it  is  possible  that  cattle 
may  be  infected  with  human  tubercle  bacilli.  Bovine  tubercle  bacilli 
are  more  virulent  for  some  animals,  as  rabbits,  than  human  tubercle 
bacilli.2  It  seems  possible  that  the  danger  of  infection  from  cattle  has 
been  somewhat  overrated. 

The  lesion  produced  by  the  tubercle  bacilli  in  the  tissues 
of  men  and  the  lower  animals  is  called  a  tubercle,  which 
in  the  beginning  is  a  grayish-white  area  about  the  size  of  a 

^Journal  Experimental  Medicine,  Vol.  TIL,  p.  451. 

2  T.  Smith,  Medical  Xe-^'s.  February  22,  1902 ;  Salmon,  Bureau  of 
Animal  Industry.  Bui.  \«>.  .^ :  Adami,  Philadelphia  Medical  Journal, 
February  _>2,  iuo_>:  Ravenel,  Unirersity  of  Pennsylvania  Medical 
bulletin.  May,  1902;  Larti.uau.  Journal  Medical  Research.  Vol.  VI.,  1901. 


PATHOGENIC    BACTERIA.  29! 

millet-seed.  In  sections  of  the  tissue  young  tubercles  are 
found  to  present  several  different  structures.  Near  the 
center,  one  or  more  very  large  cells  called  giant-cells  occur. 
They  contain  several  or  many  nuclei  which  are  frequently 
arranged  in  a  crescentic  manner  at  one  side  of  the  cell. 
Tubercle  bacilli  can  sometimes  be  demonstrated  inside  of  the 
giant-cell.  Except  possibly  in  the  very  youngest  tubercles, 
a  small  area  of  necrotic  tissue  will  always  be  found  at  the 
center  of  the  tubercle. 

Around  the  giant-cells  and  the  necrotic  area  are  seen 
large  cells  with  distinct  nuclei  which  resemble  epithelial 
cells,  and  are  often  called  epithelioid  cells ;  they  are  also 
often  termed  granulation  cells,  and  represent  an  attempt 
at  the  formation  of  granulation  tissue.  But  no  new-formed 
blood-vessels,  such  as  are  found  in  granulation  tissue  as  a 
rule,  occur  in  the  tubercle.  Tubercle  bacilli  may  also  be 
found  among  the  epithelioid  cells.  Outside  of  these  epithe- 
lioid cells  is  another  layer  of  small  cells  called  lymphoid 
cells  which  represent  leucocytes  that  have  appeared  in  this 
situation  as  a  part  of  the  inflammatory  reaction  excited  by 
the  presence  of  the  tubercles.  The  zone  of  lymphoid  cells 
may  be  very  indistinct  or  wanting.  Frequently  it  may  be 
very  difficult  to  make  out  that  the  cells  are  arranged  in 
distinct  zones  at  all.  The  cells  are  imbedded  in  a  matrix 
consisting  of  the  connective  tissue  originally  belonging  to 
the  part,  to  which  some  fibrin  may  be  added.  In  addition 
to  the  fact  that  no  new  blood-vessels  are  formed  to  main- 
tain the  nutrition  of  these  newly-formed  cells,  the  small 
vessels  included  in  the  tubercle  and  around  it  suffer  from 
inflammatory  changes.  Owing  to  these  causes  and  to  a 
toxic  substance  formed  by  or  in  the  tubercle  bacilli,  de- 
generative changes  and  necrosis  take  place  at  the  central 
part  of  the  tubercle.  As  a  result  of  these  degenerative 
changes  the  center  of  the  tubercle  becomes  converted  into 
26 


2Q2  MANUAL    OF    BACTERIOLOGY. 

a  dry,  yellowish-white,  friable  mass,  resembling  dry  cream- 
cheese.  Such  material  is  said  to  be  caseous,  and  the  pro- 
cess is  called  cascation.  Prudden  and  Hodenpyl  found  that 
the  injection  of  dead  tubercle  bacilli  into  animals  produced 
lesions  having  the  histological  characters  of  tubercles,  but 
caseation  did  not  take  place. 

The  small  tubercles  first  formed  are  called  gray  or  mili- 
ary  tubercles.  As  they  become  larger  they  also  frequently 
become  confluent.  The  larger,  confluent,  caseous  tubercles 
are  often  called  yellow  tubercles.  Swollen  tuberculous 
lymph-nodes  of  the  neck  are  among  the  manifestations  of 
the  condition  formerly  known  as  scrofula. 
""Masses  of  caseous  tubercles  sometimes  undergo  soften- 
ing. In  the  lungs  the  discharge  of  the  softened  material 
results  in  the  formation  of  a  cavity.  This  formation  of  a 
cavity  in  the  lungs  is  frequently,  if  not  usually,  accom- 
panied by  secondary  infection  with  pyogenic  micrococci. 
Caseous  tuberculous  masses  may  become  partly  calcified. 
Very  often  they  may  be  encapsulated  by  new  formed  fibrous 
or  scar  tissue.  It  is  possible  for  tuberculosis  to  become 
cured  for  all  practical  purposes  by  means  of  this  process. 
Autopsies  on  human  subjects  have  shown  that  such  cures 
not  rarely  take  place,  especially  in  tuberculosis  of  the  lungs 
occurring  over  a  localized  area.  The  statistics  of  autopsies 
vary  widely  as  to  the  number  of  persons  that  at  some  time 
of  life  suffer  from  tuberculosis  (25  or  30  per  cent,  and 
upwards).  When  a  tuberculous  area  has  become  caseous 
and  encapsulated  and  apparently  quiescent,  it  is  possible  for 
it  to  be  excited  to  renewed  activity  under  suitable  conditions, 
and,  owing  to  the  softening  and  the  discharge  of  infected 
material  into  one  of  the  vessels  or  cavities  of  the  body,  a 
wide-spreading  and  rapidly  fatal  tuberculosis  may  follow. 

Tuberculosis  may  become  disseminated  throughout  the 
body  from  a  small  focus  as  a  starting-point.     The  tubercle 


f 

PATHOGENIC    BACTERIA.  293 

bacilli  may  travel  through  the  lymph-spaces  and  affect  adja- 
cent tissues,  some  of  them  reaching  the  nearest  group  of 
lymph-nodes.  In  tuberculosis  of  the  lungs  it  is  usual  also  to 
find  tubercles  in  the  bronchial  lymph-nodes,  and  in  tuber- 
culosis of  the  intestines  there  is  also  tuberculosis  of  the 
mesenteric  lymph-nodes.  The  disease  may  travel  along  the 
serous  surfaces  and  become  widely  scattered  throughout  a 
cavity  like  that  of  the  pleura  or  peritoneum.  The  bacilli 
may  be  expelled  on  some  mucous  surface  and  be  carried 
along  it  to  infect  some  point  farther  on,  as  happens  when 
the  larynx  becomes  infected  in  tuberculosis  of  the  lungs, 
and  when  in  the  same  disease  tuberculous  sputum  is  swal- 
lowed and  leads  to  infection  of  the  intestines.  Finally,  the 
infectious  material  may  enter  the  blood-vessels,  especially 
the  veins,  and  be  swept  along  with  the  blood-current  to 
become  scattered  generally  throughout  the  body.  In  such 
cases  we  are  likely  to  have  general  or  acute  miliary  tubercu- 
losis. Almost  every  organ  of  the  human  body  may  be  in- 
fected by  tuberculosis.  Among  the  most  common  may  be 
mentioned  the  lungs,  the  lymph-nodes,  the  bones,  the  intes- 
tines, the  skin,  the  meninges,  and  the  serous  membranes. 

Infection,  as  far  as  we  know,  is  always  to  be  attributed 
directly  or  indirectly  to  some  preexisting  case  of  tubercu- 
losis in  man  or  the  lower  animals.  The  entrance  into  the 
body  is  most  commonly  by  way  of  the  lungs,  where  also 
tuberculous  disease  is  commonest  in  man,  going  by  the 
name  of  consumption.  This  is  doubtless  due  to  the  prev- 
alent habit  of  expectorating  in  public  places.  Out  of  fifty- 
six  samples  of  sputum  collected  in  street  cars  by  Dr.  W.  G. 
Bissell,  City  Bacteriologist  in  Buffalo,  four  were  tubercu- 
lous. In  forty-eight  samples  taken  from  the  floors  of  a 
public  building  by  Dr.  C.  R.  Orr,  of  the  pathological  labor- 
atory of  the  University  of  Buffalo,  tubercle  bacilli  were 
found  three  times.  According  to  the  researches  of  Nuttall, 


294  MANUAL    OF    BACTERIOLOGY. 

a  case  of  tuberculosis  may  expectorate  many  millions  of 
tubercle  bacilli  in  the  course  of  twenty-four  hours.  Cough- 
ing and  similar  efforts  may  serve  to  disseminate  the  bacilli 
(see  page  163). 

Concerning  the  occurrence  of  tubercle  bacilli  in  cow's 
milk  and  butter,  see  pages  148  and  149. 

Cases  have  been  recorded  in  which  the  disease  was  trans- 
mitted from  the  mother  to  the  child  in  the  uterus ;  how  fre- 
quently this  happens  is  uncertain.  It  is  usual  to  attribute 
greater  importance  to  an  inherited  tendency  to  tuberculosis 
than  to  the  inheritance  of  the  tubercle  bacilli  themselves. 

Agglutination  of  the  tubercle  bacillus  is  said  to  occur  with 
the  serum  of  cases  of  tuberculosis  under  certain  circum- 
stances. The  reaction  does  not  seem  likely  to  be  of  practical 
value. 

Tuberculin  is  made  by  concentrating  a  culture  of  tuber- 
cle bacilli  grown  in  glycerin-bouillon  to  one-tenth  of  its 
original  volume,  over  a  water-bath,  and  filtering  through  an 
unglazed  porcelain  filter.  It  therefore  represents  the  pro- 
ducts of  tubercle  bacilli.  It  was  proposed  by  Koch  as  a 
remedy  for  tuberculosis,  but  it  has  not  met  with  great  suc- 
cess, and  is  little  used  as  a  therapeutic  agent.  It  has  been 
found,  however,  of  great  value  in  the  diagnosis  of  tubercu- 
losis, especially  in  cattle.  When  tuberculin  is  injected  into 
a  tuberculous  animal  there  results  considerable  general  dis- 
turbance, of  which  the  most  noticeable  evidence  is  a  sud- 
den rise  in  temperature,  while  hyperemia  is  excited  around 
the  tuberculous  area.  In  a  healthy  subject  the  injection 
produces  no  reaction.  There  is  danger  attending  its  use, 
so  that  its  application  in  diagnosis  is  practically  confined  to 
cattle.1  As  a  diagnostic  measure  in  cattle  it  has  been  found 
accurate  in  the  great  majority  of  cases.  Concerning  tuber- 

1  For   details   as   to   its   use   in   cattle   see   V.    A.    Mi  tore,    "Infectious 

Diseases  <>f  Animals,"  i<)O_\  p.  151. 


PATHOGENIC    BACTERIA.  295 

culosis  in  cows,  see  page  148.  Supposing  that  some  cura- 
tive principle  exists  in  the  bodies  of  the  tubercle  bacilli 
themselves  which  could  not  be  procured  from  cultures  de- 
prived of  their  bacilli  by  nitration  through  porcelain,  Koch 
has  recently  proposed  a  new  form  of  tuberculin  called 
"  tuberculin  R,"  which  consists  of  an  extract  made  from 
dried  and  pulverized  living  tubercle  bacilli.  The  value  of 
this  new  tuberculin  as  a  remedy  is  at  least  doubtful,  and 
physicians  are  disposed  to  regard  it  with  a  great  deal  of 
caution. 

Immunity  from  tuberculosis  has  been  attained  experimentally  to  a 
certain  degree.  In  very  old  cultures  the  virulence  of  tubercle  bacilli 
sometimes  becomes  greatly  diminished.  Animals  which  survive  injec- 
tions of  such  bacilli  may  afterwards  withstand  large  doses  of  virulent 
bacilli.1 

Acid-proof  bacilli  resembling  tubercle  bacilli  have  been  alluded  to 
a  number  of  times  (pages  149,  154,  and  288).  A  number  of  such  bacilli 
have  been  cultivated,  such  as  those  of  butter  and  grass.  Injected  into 
animals  they  may  produce  nodules  more  or  less  like  tubercles.  In  these 
nodules  they  sometimes  assume  forms  resembling  the  fungus  of  actino- 
mycosis.  The  tubercle  bacillus  rarely  shows  similar  forms.  All  the 
bacilli  of  this  class,  including  the  tubercle  bacillus,  sometimes  show 
branching.  It  is  probable  that  the  bacilli  of  this  group  are  related  to 
the  fungus  of  actinomycosis.2  Similar  organisms  have  been  found  in 
fishes,  in  whom  they  produce  nodules  resembling  tubercles;  it  is  quite 
possible  that  the  latter  organisms  are  tubercle  bacilli,  which  have  been 
modified  by  an  altered  environment.  Another  acid-proof  bacillus  has 
been  found  which  is  pathogenic  to  rats,  producing  lesions  of  the  skin 
with  nodules ;  the  disease  appears  in  wild  rats  in  certain  localities. 

Tuberculosis  of  Birds. — Fowls,  ducks  and  other  birds  sometimes 
suffer  from  tuberculosis  due  to  a  bacillus  closely  resembling  the  tubercle 
bacillus  of  mammals.  It  has  similar  staining  properties.  It  sometimes 
grows  in  long,  branching  forms.  It  differs  somewhat  from  the  tubercle 
bacillus  of  mammals  in  its  cultural  properties.  The  liver  is  the  organ 
most  often  affected.  Guinea-pigs  are  much  less  susceptible  to  it  than  to 

'Truder.ti,  Neiv  York  Medical  Journal,  July  18,  1903.  Salmon,  Phila- 
delphia Medical  Journal,  June  13,  1903. 

2  Abbott  and  Gildersleeve,  University  Pennsylvania  Medical  Bulletin, 
June,  1902. 


296  MANUAL    OF    BACTERIOLOGY. 

mammalian  tuberculosis.    Rabbits  are  somewhat  susceptible,  though  less 
so  than  to  mammalian  tuberculosis. 

Pseudo-tuberculosis. — Guinea-pigs  and  other  rodents  sometimes  pre- 
sent lesions  macroscopically  very  similar  to  those  of  tuberculosis,  in 
which,  however,  the  tubercle  bacilli  cannot  be  found.  These  affections 
appear  not  to  be  tuberculosis  at  all,  and  their  nature  is  not  well  under- 
stood. Several  organisms  have  been  found  in  them,  all  of  which  are 
entirely  unlike  the  tubercle  bacillus. 

Bacillus  leprae  (of  leprosy). — A  slim  bacillus  about  4/^ 
in  length.  It  is  probably  not  motile.  It  is  uncertain  whether 
or  not  it  forms  spores.  It  stains  by  the  Gram  and  the 
Weigert  fibrin  method,  and  it  is  also  colored  by  the  methods 
used  for  staining  the  tubercle  bacillus.  It  takes  the  dye, 
however,  more  readily  than  the  tubercle  bacillus.  In  stained 
preparations  it  appears  very  similar  to  the  tubercle  bacillus, 
and  resembles  it  in  having  alternate  colored  and  unstained 
spots.  Although  several  observers  have  reported  success 
in  attempts  to  cultivate  the  bacillus  of  leprosy,  their  claims 
have  been  disputed.  The  results  of  inoculation  into  man 
and  the  lower  animals  of  material  coming  from  cases  of 
leprosy  have  also  been  uncertain.  The  bacillus  of  leprosy 
has  been  found  so  constantly  in  the  tissues  of  those  having 
the  disease  that  it  is  generally  admitted  to  be  the  specific 
cause.  The  skin  and  the  peripheral  nerves  are  the  parts 
most  affected,  although  other  tissues  and  the  internal  viscera 
may  be  involved.  A  granulation  tissue,  forming  nodules 
and  thickenings,  appears  in  the  affected  parts.  The  bacilli 
are  found  in  large  numbers  in  the  nodules,  partly  outside  of 
the  cells,  but  mostly  within  the  cells.  It  is  still  uncertain 
whether  or  not  the  disease  can  be  transmitted  directly  from 
one  individual  to  another,  in  extra -uterine  life,  or  whether 
it  can  be  inherited  from  the  parents.  However,  no  explana- 
tion can  be  given  for  the  appearance  of  the  infection  in  any 
patient,  except  communication  with  some  other  case. 
Transmission  by  contact  seems  at  any  rate  not  to  take  place 
easily. 


PATHOGENIC    BACTERIA.  297 

Bacillus  mallei  (of  glanders). — A  slim  bacillus  with 
round  or  pointed  ends,  which  often  shows  alternate  light 
and  dark  spots  in  stained  preparations.  Branching  forms 
have  been  described.  It  is  not  motile.  It  probably  does 
not  form  spores.  It  is  decolorized  by  Gram's  method. 
After  staining  with  the  ordinary  aniline  dyes  it  is  easily  de- 
colorized, and  on  that  account  it  is  difficult  to  demonstrate 
in  sections  of  tissues.  It  is  facultative  anaerobic.  It  grows 
at  the  room  temperature,  but  better  in  the  incubator.  It 
grows  slowly  on  gelatin,  and  does  not  liquefy  it,  or  only 
after  a  long  time.  On  agar  it  produces  a  moist,  white 
growth,  on  blood-serum  a  yellowish  or  brownish  growth; 
blood-serum  is  not  liquefied.  Milk  is  coagulated  slowly, 
and  the  reaction  becomes  acid.  On  potato  the  growth  is 
characteristic  in  one  or  two  days  in  the  incubator,  becom- 
ing translucent  amber-yellow,  later  a  reddish-brown,  while 
the  surface  of  the  potato  becomes  discolored. 

It  is  killed  in  five  minutes  by  a  5  per  cent,  solution  of 
carbolic  acid,  in  two  minutes  by  1-5000  bichloride  of  mer- 
cury. It  may  survive  drying  for  a  number  of  weeks. 

In  the  horse  and  ass  it  produces  the  disease  known  as 
glanders,  which  affects  the  mucous  membrane  of  the  nasal 
cavity.  When  the  skin  is  involved  the  disease  goes  by  the 
name  of  farcy.  In  the  nose,  nodules  appear  in  the  mucous 
membrane  which  become  necrotic,  forming  ulcers.  They 
may  become  confluent,  and  may  extend  along  the  adjacent 
surfaces  as  far  as  the  lungs.  There  is  a  profuse  discharge 
from  the  nose.  The  neighboring  lymph-nodes  become  in- 
volved and  are  swollen,  and  nodules  may  be  present  in  the 
internal  viscera.  In  the  skin  the  nodes  lying  underneath 
the  skin  are  called  farcy-buds.  Histologically  the  nodules 
consist  of  a  granulation  tissue,  but  they  tend  to  break  down 
rapidly,  and  the  process  in  some  respects  is  very  like  ordi- 
nary suppuration. 


298  MANUAL    OF    BACTERIOLOGY. 

This  bacillus  is  pathogenic1  to  guinea-pigs,  field-mice  and 
cats;  rabbits,  sheep  and  dogs  are  less  susceptible  or  only 
slightly  so,  also  white  and  house-mice,  and  hogs ;  cattle  are 
immune.  Men  are  occasionally  affected,  especially  those 
who  have  come  in  contact  with  horses.  The  mucous  mem- 
brane of  the  nasal  cavity  may  be  the  part  involved,  or  the 
skin,  or  the  internal  viscera.  In  a  number  of  instances, 
workers  in  the  laboratory  have  been  accidentally  infected. 

The  diagnosis  of  the  disease  is  best  effected  by  the  in- 
oculation of  a  male  guinea-pig  with  the  material  from  a 
case  suspected  of  being  glanders,  introducing  it  into  the 
peritoneal  cavity  (Method  of  Straus).  In  about  two  to 
three  days  there  appears  a  characteristic  swelling  of  the 
testicle  indicating  the  beginning  of  suppuration,  which 
presently  takes  place ;  the  animal  usually  dies  after  two  or 
more  weeks.  At  least  two  guinea-pigs  should  be  inocu- 
lated: and  the  test  may  sometimes  fail,  when  it  should  be 
repeated  on  other  guinea-pigs.2 

Mallcin  is  a  product  obtained  from  an  old  glycerin- 
bouillon  culture  of  the  bacillus  mallei.  The  cultures  are 
placed  in  a  steam  sterilizer  for  several  hours,  and  are  fil- 
tered through  unglazed  porcelain.  The  filtrate  contains 
the  products  of  the  growth  of  the  bacillus  mallei  and  is  of 
much  the  same  character  as  tuberculin.  Injected  into  ani- 
mals suspected  of  having  glanders,  if  it  produces  a  local 
and  febrile  reaction,  the  existence  of  glanders  is  indicated. 
It  is  usually  successful  in  the  diagnosis  of  the  disease  in 
lower  animals,  especially  in  horses,  where  it  has  been 
largely  employed.  An  agglutination  reaction  has  been  de- 
scribed for  the  bacillus  of  glanders. 

1The  statements  of  different   writers  differ  considerably   \\ilh  regard 
to  some  of  these  animals. 
2  Frothingham,  Journal  Medical  Research.  Vol.  VI.,  1901. 


PATHOGENIC    BACTERIA. 


299 


Actinomyces  bovis1  ( Streptothrix  actinomyces,  Ray- fun- 
gus of  actinomycosis). — The  morphology  of  this  organism 
is  quite  different  from  that  of  most  of  the  bacteria.  It  is 
sometimes  considered  to  be  a  bacterium  of  a  higher  type. 
The  organism  appears  in  the  form  of  threads  which  show 
genuine  branching.  These  threads  make  radiating,  inter- 
lacing masses.  Their  external  ends  are  swollen  and  bulbous 
under  certain  conditions.  Colonies  formed  in  this  manner, 
seen  under  moderate  mag- 
nification, have  a  radiat- 
ing appearance  which  has 
given  rise  to  the  name, 
ray-fungus.  The  club- 
shaped  external  ends  are 
readily  distinguished  and 
the  growth  possesses  a 
very  distinctive  form. 
This  is  the  shape  which 
the  organism  presents  as 
it  grows  in  the  animal 
body.  The  club-shaped 
ends  are  generally  re- 

1     i  i  Ray-fungus  of  Actinomycosis.      Fresh, 

garded     as     a     degener- 

0  °  unstained  preparation  from  a  case  of 

ative    Or    involution    form.  lump.jaw  in  a  cow.      Diagrammatic. 

Transverse  divisions  may 

sometimes  be  distinguished  upon  the  threads.  Spherical 
forms  resembling  micrococci  may  appear  which  may  pos- 
sibly be  spores.  In  some  members  of  this  group  spores 
form  in  cultures  on  the  ends  of  the  filaments  (conidia). 
The  organism  stains  with  the  ordinary  aniline  dyes,  by 
Gram's  method  or  the  Weigert  fibrin  stain. 

The  fungus  may  be  cultivated  upon  the  usual  culture- 
media,  though  not  easily.     It  is  facultative  anaerobic.     It 

1  Hektoen,  Philadelphia  Monthly  Medical  Journal,   November,    1899; 
Ewing,  Bulletin  Johns  Hopkins  Hospital,  November,  1902. 


300 


MANUAL    OF    BACTERIOLOGY. 


grows  both  at  ordinary  temperatures  and  in  the  incubator. 
The  growth  is  not  rapid.  The  colonies  are  fine,  dry, 
elevated,  irregular  in  form,  becoming  opaque.  Bulbous 
ends  upon  the  threads  do  not  usually  appear  in  cultures. 
The  results  of  the  injection  of  these  cultures  into  the  lower 
animals  are  as  yet  uncertain. 

The  disease  produced  by  the  ray-fungus  is  called  actino- 
mycosis.     It  occurs  in  cattle  chiefly,  seldom  in  swine  and 

FIG.  84. 


Actinomyces  bovis,  smear  preparation   from   a   pure  culture,   stained  by 
Gram's  method.     (X  1000.) 

horses,  and  occasionally  in  man.  Infection  appears  to  be 
carried  by  grain  or  particles  of  vegetable  fiber  which  pene- 
trate the  tissue.  The  presence  of  such  foreign  particles  as 
well  as  the  organism  appears  to  favor  infection.  The  infec- 
tious material  frequently  enters  through  the  mouth,  espe- 
cially in  the  vicinity  of  the  teeth,  but  it  may  also  occur 
through  the  skin  or  the  mucous  membranes.  It  leads  to  the 


PATHOGENIC    BACTERIA.  3OI 

formation  of  inflammatory,  tumor-like  nodules,  hence  the 
name  "  lump-jaw  "  given  to  the  disease  in  cattle.  Necrosis 
of  the  tissue  takes  place  with  the  formation  of  an  abscess. 
The  pus  is  peculiar  in  containing  small  whitish  particles 
which  consist  of  little  colonies  of  the  ray-fungus,  and  which 
readily  permit  the  disease  to  be  diagnosed  by  the  micro- 
scope. The  material  may  be  examined  in  the  perfectly 
fresh  condition  without  any  staining.  The  jaw  or  its  neigh- 
borhood is  very  frequently  affected,  or  the  disease  may  be 
present  in  other  situations  about  the  head  and  neck,  and 
may  involve  the  lungs,  the  intestines,  and  the  vertebrae,  ribs, 
and  other  bones.  The  disease  is  usually  localized,  but  a 
number  of  areas  may  be  affected  simultaneously. 

Besides  the  common  actinomyces,  there  are  numerous  other  ray-fungi, 
more  or  less  closely  related,  and  whose  pathogenic  properties  are  not 
fully  determined.  Generally  speaking  they  appear  to  be  saprophytes 
naturally,  which  occasionally  become  parasitic  and  pathogenic  under 
especially  favorable  conditions.  A  number  of  species  have  been  found 
in  air,  dust,  etc.,  some  of  them  chromogenic.  Wolff  and  Israel  described 
an  anaerobic  species,  pathogenic  to  man  and  animals.  Madura  disease, 
Madura  foot,  or  mycetoma  is  a  disease  occurring  in  India  (rarely  else- 
where), affecting  one  of  the  extremities,  characterized  by  swellings, 
nodular  deposits  and  abscesses.  Some  cases  are  certainly  due  to  a  mem- 
ber of  the  actinomyces  group.1 

Other  branching  organisms,  some  of  them  acid-proof,  have  been  de- 
scribed chiefly  under  the  name  of  streptothrix.  In  man  they  have  been 
found  in  a  variety  of  suppurative  and  necrotic  lesions,  in  particular, 
broncho-pneumonias.2 

Bacillus  iyphosus  (Bacillus  of  Eberth). — A  bacillus 
with  rounded  ends,  varying  in  length,  sometimes  making 
very  short,  oval  forms,  sometimes  growing  out  into  long 
threads.  It  is  very  actively  motile,  and  possesses  numerous 

1  Compare  Wright,  Journal  Experimental  Medicine,  Vol.  III.,  p.  421. 

2  Norris  and  Larkin,  Journal  Experimental  Medicine,  Vol.  V.,  p.  155; 
Musser,    Philadelphia    Medical    Journal,    September    7,    1901;    Flexner, 
Journal  Experimental  Medicine,  Vol.   III.;   MacCallum,   Centralblatt  f. 
Bakteriologie,  Orig.  Bd.  XXXI.,  1902. 


3O2  MANUAL    OF    BACTERIOLOGY. 

flagella  which  arise  from  all  parts  of  the  surface.  It  does 
not  form  spores.  It  is  not  stained  by  Gram's  method,  but 
it  may  be  colored  with  the  ordinary  aniline  dyes,  when  the 
stain  will  frequently  be  somewhat  irregular.  It  may  be 
stained  in  sections  of  tissues  from  cases  of  typhoid  fever, 
with  the  aniline  dyes,  such  as  Loffler's  alkaline  methylene- 
blue.  It  is  facultative  anaerobic.  It  grows  at  ordinary 
temperatures,  better  in  the  incubator,  but  grows  rather  more 
slowly  than  B.  coli  commnnis.  Gelatin  is  not  liquefied. 


X, 

-  ^   /  I 

•"*  '-*. 


'"• 


>•/»•    \ 
'  *N\   1% 

Bacillus   of  typhoid   fever.      (X  n 

surface  colonies  in  gelatin  appear  whitish,  with 
irregular  borders  and  more  or  less  wrinkled  surfaces,  when 
slightly  magnified.  It  grows  on  the  ordinary  media,  and  the 
growths  are  whitish.  Bouillon  is  clouded.  Milk  becomes 
slightly  acid,  but  is  not  coagulated.  In  media  containing 
dextrose,  acid  is  formed  but  no  gas.  In  lactose-bouillon 
neither  acid  nor  gas  is  formed,  although  when  grown  in 
milk  the  typhoid  bacilli  produce  an  acid  reaction.  The  lac- 


PATHOGENIC    BACTERIA. 


303 


tose-litmus-gelatin  or  -agar  of  VVurtz  makes  use  of  the  blue 
tinge  possessed  by  colonies  of  the  typhoid  bacillus  on  this 
medium  to  distinguish  them  from  colonies  of  the  colon 
bacillus  and  other  bacteria  which  form  acids  from  lactose. 
Neutral  red  has  been  used  in  the  same  manner,  as  it  is  said 
not  to  be  altered  by  the  typhoid  bacillus,  but  to  be  changed 
by  the  colon  bacillus  to  a  yellow  color.  (To  neutral,  plain 
agar  add  i  per  cent,  of  a  saturated  aqueous  solution  of 
neutral  red,  and  some  also  add  .3  per  cent,  dextrose.) 

FIG.  86. 


• 


' 

' 

, 

-K&- 


i     ' 

•~f- 


Bacillus  of  typhoid  fever,  stained  by  Loffler's  method  to  show  flagella. 

(X  1000.) 

Ill  Dunham's  peptone  solution  indol  is  not  formed,  as  a 
rule.  On  potato  it  usually  forms  what  is  called  an  invisible 
growth;  that  is,  although  no  development  is  apparent  to  the 
eye,  numerous  bacilli  may  be  shown  under  the  microscope 
in  smear  preparations  made  from  the  surface  of  potato 
inoculated  about  forty-eight  hours  previously.  Occasionally 
a  slight  visible  growth  is  seen  on  potato. 


3O4  MANUAL    OF    BACTERIOLOGY. 

The  typhoid  bacillus  is  killed  at  60°  C.  in  ten  minutes. 
It  resists  drying  well.  It  can  survive  in  soil  and  sewage  a 
long  time. 

For  a  comparison  of  the  properties  of  the  typhoid  bacillus 
and  the  colon  bacillus  see  the  latter. 

Concerning  the  detection  of  the  typhoid  bacillus  in 
water  see  page  142. 

A  new  medium  has  been  suggested  by  Hiss1  for  the  isolation  of  the 
typhoid  bacillus.  It  consists  of  gelatin  and  agar,  beef-extract,  sodium 
chloride  and  dextrose,  and  is  given  a  slightly  acid  reaction.  These  sub- 
stances are  used  in  different  proportions  for  plate-  and  for  tube-cul- 
tures. This  medium  is  of  a  semi-solid  character,  and  makes  use  of  the 
great  motility  of  the  typhoid  bacillus  in  producing  a  uniform  clouding 
of  the  medium  in  tubes,  with  the  absence  of  a  gas  formation;  while 
in  plate-cultures  the  colonies  exhibit  peculiar  filamentous  outgrowths. 
It  is  claimed  that  it  can  be  determined  whether  organisms  are  typhoid 
bacilli  or  not  after  thirty-six  hours  in  the  incubator. 

Other  special  media  for  the  identification  of  the  typhoid  bacillus  have 
been  devised  by  Eisner,  Stoddart,  by  Capaldi  and  Proskauer,  and  bj 
Piorkowski.2  The  medium  of  Stoddart  is  based  upon  principles  similar 
to  those  applied  in  the  medium  of  Hiss. 

M.  W.  Richardson  has  devised  an  application  of  the  serum-test  to 
plate-colonies  suspected  of  containing  typhoid  bacilli.  If  a  typhoid  colony 
be  torn  with  a  needle,  under  moderate  magnification  "  a  seething  motion 
resembling  much  the  appearance  of  a  swarm  of  bees "  may  be  seen. 
This  appearance  is  due  to  the  motility  of  the  bacteria.  If  such  a  colony 
be  touched  with  a  small  quantity  of  blood-serum  from  a  case  of  typhoid 
fever,  the  motion  is  said  to  cease  instantly  and  almost  absolutely. 
Colonies  of  other  motile  bacteria  do  not  undergo  a  corresponding  loss 
of  motility. 

THE  SERUM-TEST  FOR  TYPHOID  FEVER.3 
When  a  small  quantity  of  a  culture  of  typhoid  bacilli  is 
mixed  with  a  little  blood-serum  derived  from  a  case  of  ty- 

1  Journal  Medical  Research,  Vol.  VIII.,  1002. 

"Eisner,  Zcitschrift  f.  Hygiene,  Bd.  XXL,  1895,  p.  25;  Stoddart. 
Journal  of  Pathology  and  Bacteriology,  Vol.  IV.,  p.  429,  1897;  Capaldi 
and  Proskauer,  Zcitschrift  fiir  Hygiene,  etc.,  Bd.  XXIII. ,  p.  452,  1896; 
Piorkowski,  Berliner  klinischc  irochcnschrift,  1899,  p.  145. 

3  This  test  is  often  known  as  the  "  Widal  reaction."  For  a  history  and 
general  discussion  of  the  subject  see  Durham,  Journal  of  Experimental 
Medicine,  Vol.  V.,  p.  353. 


PATHOGENIC    BACTERIA. 


305 


phoid  fever,  within  a  few  minutes  the  motility  of  the  typhoid 
bacilli  is  abolished  and  they  become  agglutinated  into 
clumps  or  masses.  Occasionally  the  bacilli  may  eventually 
undergo  disintegration  into  granular  material.  This  re- 
action does  not  take  place  with  the  blood-serum  of  healthy 
persons  or  of  those  suffering  with  other  diseases,  nor  when 
the  blood-serum  of  a  typhoid  fever  case  is  mixed  with  motile 
bacteria  other  than  typhoid  bacilli.  It  has  been  observed 
in  the  blood-serum  of  an  infant  born  while  the  mother  was 
convalescing  from  typhoid  fever. 

FIG.  87. 


Application  of  the  serum-reaction  to  typhoid  bacilli.  A  shows  the  dis- 
tribution of  the  bacilli  before  the  reaction.  It  is  to  be  remembered  that 
they  are  motile  and  their  positions  may  change  continually.  B  shows 
clumping  of  the  motionless  bacilli  after  mixture  with  the  serum  of  a  case 
of  typhoid  fever.  Diagrammatic. 

The  agglutinating  substance  has  been  found  in  blister- 
serum  and  in  the  milk  of  typhoid  cases,  in  fluids  from  the 
serous  cavities  and  inflammatory  and  edematous  areas  in 
variable  amounts,  and  occasionally  in  urine,  bile  and  tears. 

The  reaction  may  be  obtained  by  adding  blood-serum  to 
a  young  bouillon-culture  of  typhoid  bacilli  kept  in  the  in- 
cubator, when  the  occurrence  of  agglutination  becomes 


306  MANUAL    OF    BACTERIOLOGY. 

manifest  by  the  collection  of  the  bacteria  into  visible  masses 
or  flocculi,  which  form  a  sediment.  Most  investigators 
prefer  to  watch  the  results  under  the  microscope,  using  an 
ordinary  slide,  or,  better,  the  hanging-drop.  Young  cultures 
—less  than  twenty-four  hours  old — in  bouillon,  and  kept  in 
the  incubator,  may  be  used,  or  better  cultures  kept  at  room- 
temperature  for  twenty-four  hours.  Johnston  and  Mc- 
Taggart  recommend  that  the  bouillon  cultures  be  freshly 
made  each  time  from  stock  cultures  on  agar,  which  need  only 
occasionally  be  transplanted.  Certain  stocks  of  typhoid 
bacilli  seem  especially  suited  to  this  reaction,  and  such  a 
stock  should  be  secured. 

Blood-serum,  blister-serum,  fresh  blood  and  dried  blood 
have  all  been  tried  with  success.  Blood  dried  on  unglazed 
paper  or  cover-glasses  as  proposed  by  Wyatt  Johnston  is  ex- 
tremely convenient.  To  perform  the  test,  it  is  mixed  with 
sterilized  distilled  water,  bouillon,  or  normal  salt  solution; 
the  objection  to  it  lies  in  the  difficulty  of  securing  an  accu- 
rate dilution.  An  approximate  knowledge  of  the  degree  of 
dilution  may  be  acquired  by  mixing  drops  of  dried  blood 
of  known  volume  with  definite  amounts  of  water,  and  ob- 
serving the  tints.  These  should  be  kept  in  mind  as  stand- 
ards. The  dilution  may  be  measured  with  the  hemoglo- 
1)inometer  or  with  the  pipette  of  the  hemacytometer.  The 
Xe\v  York  Board  of  Health  have  found  blister-serum  satis- 
factory and  easy  to  obtain.  A  little  of  the  diluted  serum  is 
mixed  on  the  cover-glass  with  a  definite  amount  of  the  fresh 
bouillon-culture,  and  is  examined  as  a  hanging-drop.  In 
a  short  time  the  characteristic  clumping  and  loss  of  motility 
occur.  At  the  same  time  a  drop  of  the  culture  alone,  and 
a  drop  of  the  culture  mixed  with  normal  scrum-,  similarly 
diluted,  should  be  examined  as  controls.  The  dilutions 
used  vary  from  I  part  of  serum  in  30  to  I  in  50.  The 
higher  dilutions  are  more  accurate.  The  time  within  which 


PATHOGENIC    BACTERIA.  307 

the  reaction  occurs  varies  from  a  few  minutes  to  one  or  two 
hours.  With  little  dilution  the  time  should  be  short;  with 
greater  dilution  it  may  be  longer.  Both  clumping  and 
paralysis  of  motility  should  take  place.  In  a  positive  case 
the  reaction  should  be  distinct.  Normal  blood  sometimes 
exhibits  agglutinative  properties  in  some  degree.  If  the 
reaction  in  any  case  is  not  satisfactory  it  should  be  tried 
with  a  higher  dilution,  i  to  50,  and  the  result  should  be 
positive  if  the  case  is  a  genuine  case  of  typhoid  fever. 

The  reaction  usually  appears  between  the  seventh  day 
and  the  end  of  the  third  week  of  the  disease;  it  may  be 
seen  earlier;  it  is  often  delayed  and  appears  late.  The 
test  frequently  has  to  be  repeated  when  the  first  result  is 
doubtful  or  negative.  Reports  indicate  that  the  method  is 
a  great  aid  in  the  diagnosis  of  typhoid  fever,  though  not 
infallible. 

Considerable  experience  is  necessary  to  acquire  the 
judgment  needed  in  using  this  test. 

The  agglutinating  power  becomes  lessened  after  recovery, 
and  usually  is  wanting  at  the  end  of  a  year.  Rarely  it  may 
be  present  for  a  longer  time,  a  fact  that  is  to  be  borne  in 
mind  in  diagnosis. 

Typhoid  bacilli  have  frequently  been  obtained  from  the 
stools  of  cases  of  the  disease,  but  they  are  isolated  only  with 
considerable  difficulty.  At  autopsies  they  are  best  culti- 
vated from  the  spleen,  in  which,  however,  it  is  to  be  remem- 
bered, the  bacillus  coli  communis  may  also  be  present. 
Puncture  of  the  spleen  with  a  sterilized  hypodermic  needle, 
during  life,  has  also  been  resorted  to  as  a  means  of  diag- 
nosis. The  drop  of  fluid  withdrawn  may  be  examined  by 
culture-methods  for  typhoid  bacilli.  There  is  probably  some 
clanger  to  the  patient  attending  this  procedure.  Cultures 
made  from  the  blood,  where  several  c.c.  are  taken  show  that 
a  few  bacilli  occur  in  the  blood  in  a  large  proportion  of  cases 
27 


308  MANUAL    OF    BACTERIOLOGY. 

of  the  disease,  and  probably  in  a  majority.  Typhoid  bacilli 
frequently  appear  in  the  urine  (in  about  twenty  per  cent, 
of  all  cases)  and  the  examination  of  urine  for  them  has 
been  used  in  diagnosis.  The  bacilli  often  occur  in  the 
gall-bladder.  They  have  been  found  associated  with  gall- 
stones, and  have  been  supposed  to  be  one  of  the  causes 
for  the  formation  of  gall-stones.1  They  may  remain  pres- 
ent in  the  gall-bladder  or  in  the  urine2  long  after  convales- 
cence from  the  disease.  They  have  been  demonstrated  in 
the  "  rose  spots  "  on  the  abdomen.  They  may  be  present 
in  the  lesions  of  the  pneumonia,  which  frequently  compli- 
cates typhoid  fever,  and  may  appear  in  the  sputum. 

Inoculation  experiments  in  animals  have  not  been  very 
satisfactory.  With  a  few  exceptions,  possibly,  anatomical 
lesions  resembling  those  of  typhoid  fever  have  not  been 
produced  by  the  inoculation  of  typhoid  bacilli  into  animals. 
The  injection  of  cultures  into  animals  may  produce  death, 
but  it  can  usually  be  shown  to  have  resulted  from  the  poisons 
contained  in  the  cultures. 

Typhoid  fever  is  rare  during  the  first  two  years  of  life. 
It  frequently  attacks  young  and  robust  men.  The  causes 
that  bring  about  susceptibility  to  infection  are  not  known. 

The  principal  lesion  in  typhoid  fever  lies  in  the  Peyer's 
patches  of  the  lower  part  of  the  small  intestines;  the  mes- 
enteric  lymph-nodes  and  spleen  also  are  swollen.  The 
typhoid  bacillus  may  be  demonstrated  in  sections  of  the 
walls  of  the  diseased  portions  of  the  intestine.  Cases  are 
recorded  in  which  no  lesions  were  found  in  the  intestines 
but  where  the  typhoid  bacilli  were  widely  spread  through 
the  organs  of  the  body,  and  which  therefore  represented 
typhoid  septicemia. 

Periostitis  and  osteomyelitis,  which  are  not  uncommon 
sequelae  of  typhoid  fever,  may  be  caused  by  typhoid  bacilli. 

1  Pratt,  American  Journal  Medical  Sciences,  Vol.   122,   1901. 

2  M.  \Y.  Richardson,  Journal  Experimental  Medicine,  Vol.  IV.,  1899. 


PATHOGENIC    BACTERIA.  309 

Ordinary  suppuration  may  be  produced  by  the  typhoid 
bacillus,  but  most  suppurative  affections  during  or  following 
typhoid  fever  are  mixed  infections,  or  are  due  to  the  ordi- 
nary pyogenic  bacteria. 

Typhoid  fever  is  transmitted  chiefly  through  the  medium 
of  water.  It  is  sometimes  conveyed  by  milk,  green  vege- 
tables and  oysters.  Infection  through  the  medium  of  dust, 
and  by  the  hands  and  clothing  probably  occurs,  but  not  com- 
monly. Under  certain  circumstances  the  bacilli  may  be 
carried  by  flies.1  In  caring  for  cases  of  typhoid  fever  the 
stools,  urine,  sputum  and  linen  should  be  disinfected.  Per- 
sons handling  the  patient  should  wash  and  disinfect  their 
hands. 

The  injection  of  typhoid  bacilli  which  have  been  killed  by  heat  has 
been  advised  (by  Wright)  as  a  preventive  measure.  The  results  appear 
to  have  been  partially  successful,  but  the  method  is  still  being  actively 
studied. 

Bacillus  coli  communis  (Bacterium  coli  commune  of 
Escherich,  probably  the  same  as  Bacillus  Neapolitanus  of 
Emmerich,  often  called  simply  the  colon  bacillus.  Passet 
described  an  organism  under  the  name  of  Bacillus  pyo- 
genes  fcetidus,  from  .foul  pus  and  mixed  infections,  which 
is  probably  the  same  as  B.  coli  communis). — A  bacillus  with 
rounded  ends,  frequently  of  a  short,  oval  form,  when  it 
may  be  difficult  to  distinguish  from  micrococci ;  often 
longer;  often  forming  threads.  It  is  slightly  motile,  hav- 
ing several  flagella.  It  does  not  form  spores.  It  stains 
with  the  ordinary  aniline  dyes  and  is  decolorized  by  Gram's 
method.  It  is  facultative  anaerobic.  It  grows  well  at  the 
room  temperature,  but  more  rapidly  in  the  incubator.  It 
does  not  liquefy  gelatin.  In  gelatin  plates  the  surface 
colonies  are  of  a  bluish-white  color;  the  centers  are  denser 

1  Vaughan,  Philadelphia  Medical  Journal,  June  9,  1900. 


3IO  MANUAL    OF    BACTERIOLOGY. 

than  the  borders,  which  are  translucent.  It  usually  grows 
more  rapidly  in  gelatin  than  the  bacillus  of  typhoid  fever. 
Its  growths  in  other  media  are  mostly  whitish.  Bouillon 
becomes  clouded.  Nitrates  are  reduced  to  nitrites.  In 
peptone  solution  it  forms  indol.  On  potato  it  forms  an 
abundant  visible  growth  from  cream-color  to  pale  brown. 

FIG.  88. 


v>  P* 

I*         '    ~  " 

%  \     *        0      »'.\  ^     !»       - 


v 

* 


Bacillus  coli  comraunis.     (X  1000.) 

Milk  becomes  acid  and  is  usually,  but  not  always,  coagu- 
lated slowly.  It  causes  the  development  of  gas  and  acid 
in  media  containing  dextrose  or  lactose.  In  media  contain- 
ing neutral  red  it  is  stated  that  the  colon  bacillus  produces  a 
yellow  color  with  a  green  fluorescence.  Differential  points 
between  the  bacillus  of  typhoid  fever  and  the  bacillus  coli 
communis  are  as  follows  : 

1st.  The  typhoid  bacillus  is  actively  motile;  the  •  colon 
bacillus  less  actively,  or  slightly  motile. 

2d.  The  typhoid  bacillus  has  numerous  flagella  which 
rise  from  all  parts  of  the  surface;  the  colon  bacillus  has  a 
smaller  number  of  flagella. 


f 

PATHOGENIC    BACTERIA.  311 

3d.  In  both,  spore  formation  is  absent. 
4th.  Both  are  decolorized  by  Gram's  method. 
5th.  The  colonies  of  the  typhoid  bacillus  in  gelatin  de- 
velop more  slowly  than  those  of  the  colon  bacillus. 

6th.  The   appearance   of   superficial    colonies    in    gelatin 

plates. 

FIG.  89. 


Bacillus  coli  communis  with  flagella,  stained  by  Van   Ermengem's 
method.      (X  1000.) 

7th.  In  media  containing  dextrose  or  lactose,  the  typhoid 
bacillus  does  not  produce  fermentation  with  gas  and  the 
colon  bacillus  does  produce  gas. 

8th.  The  typhoid  bacillus  produces  an  acid  reaction  with- 
out coagulation  in  milk,  and  the  colon  bacillus  produces  an 
acid  reaction  and  coagulation. 

9th.  In  peptone  solution  the  typhoid  bacillus,  as  a  rule, 
produces  no  indol ,  and  the  colon  bacillus  produces  indol. 

loth.  The  typhoid  bacillus  usually  produces  an  invisible 
growth  on  potato,  the  colon  bacillus  a  visible  growth. 

nth.  The  typhoid  bacillus  is  said  not  to  reduce  neutral 


312  MANUAL   OF    BACTERIOLOGY. 

red  in  media,  and  the  colon  bacillus  to  change  it  to  a  yellow 
color. 

To  these  may  be  added  the  growth  of  the  two  organisms 
on  special  media  like  those  of  Wurtz,  of  Eisner  and  of 
Hiss,  and  the  application  of  the  serum-reaction. 

Injections  of  cultures  of  the  bacillus  coli  communis  into 
animals  produce  variable  and  uncertain  results.  Subcuta- 
neous injection  may  lead  to  pus-formation ;  in  rabbits  and 
guinea-pigs  injections  may  produce  death  apparently  from 
poisons  introduced.  With  the  blood  of  immunized  animals 
a  serum-reaction,  similar  to  that  described  for  typhoid  fever, 
may  be  demonstrated. 

Concerning  the  occurrence  of  the  bacillus  coli  communis 
in  the  intestine  of  man,  see  page  155.* 

At  autopsies  on  human  subjects  the  great  viscera  are 
often  found  to  have  been  infected  by  the  colon  bacillus, 
usually  when  some  lesion  of  the  intestine  existed  simul- 
taneously, but  in  most  cases  without  having  produced  much 
apparent  damage  to  the  organs  invaded.  The  bacillus  coli 
communis  frequently  occurs  in  mixed  infections,  as  in 
wounds,  inflammations  and  abscesses.  It  is  often  found  in 
the  peritoneum  in  peritonitis,  in  the  pus  in  appendicitis, 
and  in  the  urine  in  cystitis;  it  frequently  occurs  in  the  in- 
terior of  gall-stones  with  whose  formation  it  may  be  con- 
nected.2 

There  is  a  large  number  of  more  or  less  closely-related 
organisms  which  go  by  the  name  of  the  "colon  group." 
The  limits  of  the  colon  group  are  extremely  ill-defined. 

Paracolon  or  paratyphoid  bacilli  are  the  names  applied  to  certain 
members  of  the  colon  group  which  have  recently  been  shown  to  be 
pathogenic  to  man.  They  may  produce  clinical  symptoms  resembling 
typhoid  fever  of  a  mild  and  atypical  form.  The  affection  is  rarely 

1  See  also   Moore  and   \Yright,   "  Bacillus   coli   communis   in   the  Do- 
niesticnu-d    Animals."  American   Medicine,   March   .'<).    K;O-'. 

2  Lartigau,  Journal  American   Medical  Association,  April   12,   1902. 


PATHOGENIC    BACTERIA.  313 

fatal.  Probably  they  may  occur  with  typhoid  fever  in  mixed  and  second- 
ary infections.  Characteristic  lesions  have  not  yet  been  observed.  The 
bacilli  have  been  found  in  the  blood,  spleen,  liver,  gall-bladder  and 
urine.  Like  typhoid  and  colon  bacilli  they  are  motile,  have  flagella,  are 
not  stained  by  Gram's  method  and  do  not  liquefy  gelatin.  They  ferment 
dextrose  and  maltose,  producing  acid  and  gas.  They  do  not  ferment 
lactose.  Milk  at  first  becomes  acid,  later  it  becomes  alkaline,  and  is  not 
coagulated.  On  potato  a  slight  visible  growth  occurs.  Media  contain- 
ing neutral  red  become  yellow,  as  with  B.  coli  communis,  but  more 
slowly,  and  the  red  color  sometimes  returns.  In  respect  to  the  fermen- 
tation of  saccharose  and  the  formation  of  indol  reports  differ;  both  are 
usually  negative.  The  blood  of  the  patient  agglutinates  the  bacilli.  But, 
as  among  the  closely  related  members  of  this  group  mutual  reactions  are 
sometimes  seen,  this  test  is  not  to  be  considered  invariable.1  Several 
bacilli  allied  to  the  above  are  known.  The  bacillus  enteridis  of  Gaertner 
is  a  related  form  which  has  been  found  in  cases  of  meat-poisoning. 

Bacillus  lactis  aerogenes  (Bacillus  aerogenes). — A  ba- 
cillus having  a  form  similar  to  that  of  the  colon  bacillus, 
described  as  being  larger  and  plumper.  In  the  main  its 
properties  are  similar  to  those  of  the  colon  bacillus.  Its 
colonies  are  more  circumscribed  and  elevated.  It  is  also 
non-motile.  It  coagulates  milk  more  rapidly  than  the 
colon  bacillus.  It  produces  gas  upon  potato  more  rapidly 
than  the  colon  bacillus,  and  more  abundantly.  It  was  de- 
scribed by  Escherich,  who  also  described  the  colon  bacillus, 
assigning  the  bacillus  lactis  aerogenes  rather  to  the  upper 
part  of  the  small  intestine,  and  the  colon  bacillus  to  the 
lower  portion.  According  to  Kruse,  the  bacillus  lactis  aero- 
genes and  its  relatives  differ  from  the  bacillus  coli  com- 
munis chiefly  in  lacking  motility.  Like  the  colon  bacillus 
it  has  been  found  many  times  in  the  urine  in  cystitis.  See 
also  B.  acidi  lactici,  page  227. 

Bacillus  dysenteriae  (Shiga). — A  bacillus  with  rounded 
ends,  of  the  size  and  shape  of  typhoid  and  colon  bacilli, 

'Gushing,  Bulletin  Jo/ins  Hopkins  Hospital,  July-August,  1900; 
Strong,  Ibid.,  May,  1902;  Johnstone,  Hewlett,  Longcope,  American 
Journal  Medical  Sciences.  August,  1902;  Libman,  Buxton,  Journal 
Medical  Researcli.  Vol.  VIII.,  1902. 


314  MANUAL    OF    BACTERIOLOGY. 

seldom  forming  threads.  Most  observers  have  found  it  non- 
motile.  Vedder  and  Duval  have  demonstrated  flagella.  The 
bacillus  does  not  form  spores.  It  may  be  stained  with  the 
ordinary  aniline  dyes;  it  does  not  stain  by  Gram's  method. 
It  is  facultative  anaerobic.  It  grows  at  ordinary  tempera- 
tures, but  better  in  the  incubator.  It  grows  on  the  usual 
culture-media,  but  more  slowly  than  B.  coli  communis. 
The  growths  are  whitish.  Colonies  on  gelatin  plates  re- 
semble those  of  the  typhoid  bacillus.  Bouillon  is  diffusely 
clouded ;  a  precipitate  may  form,  but  no  pellicle.  Indol  is 
not  produced.  Milk  becomes  acid  and  is  not  coagulated. 
On  potato  a  thin  pale  layer  forms  which  may  become  light 
brown.  No  gas  is  formed  in  media  containing  glucose  or 
lactose. 

Xeutral-red  agar  is  not  changed.  From  the  feces  the 
bacillus  is  best  cultivated  on  agar  plates,  in  the  incubator. 
Colonies  of  B.  coli  communis  are  often  more  numerous 
than  those  of  the  dysentery  bacillus.  The  colonies  which 
develop  in  twenty-four  hours  are  likely  to  be  colonies  of 
B.  coli  communis.  Their  position  may  be  marked  on  the 
glass  with  a  pencil.  Those  which  appear  later  are  to  be 
planted  in  dextrose-agar.  If  gas  develops  they  are  not  the 
bacillus  of  dysentery;  otherwise  they  are  to  be  studied  and 
identified  by  the  cultural  and  other  tests  mentioned  above, 
and  by  the  agglutination  reaction. 

The  bacilli  are  destroyed  in  a  few  minutes  by  boiling,  and 
at  58°  C.  in  half  an  hour.  They  appear  not  to  be  particularly 
resistant  to  the  influences  that  are  harmful  to  bacteria  in 
general. 

They  have  been  found  in  the  intestine  and  the  discharges 
of  acute  and  epidemic  dysentery  in  various  climates  and 
countries,  including  the  United  States.  Thus  far  their  dis- 
semination in  the  blood  and  distant  organs  has  not  been 
demonstrated.  The  lesion  of  this  form  of  dysentery  con- 


PATHOGENIC    BACTERIA.  315 

sists  of  a  severe  acute  inflammation  of  the  colon,  frequently 
with  necrosis  of  the  surface  and  the  formation  of  pseudo- 
membrane.  Ulceration  may  occur,  but  is  usually  superficial. 
Duval  and  Bassett  found  the  bacillus  of  dysentery  in  the 
stools  of  infants  having  summer  diarrhoea. 

The  introduction  of  pure  cultures  into  animals  by  way  of 
the  alimentary  canal  has  sometimes  been  followed  by  a  cer- 
tain amount  of  diarrhoea,  but  it  does  not  appear  that  dysen- 
tery, as  it  occurs  in  man,  has  been  reproduced.  Most  labor- 
atory animals  are,  however,  very  sensitive  to  the  injection 
into  the  tissues  or  veins  of  cultures,  living  or  dead.  They 
show  the  lesions  produced  by  many  toxins. 

The  bacillus  is  agglutinated  by  the  patient's  blood,  but 
often  only  late  in  the  disease  and  apparently  not  in  all  cases. 
This  test  seems  to  have  only  a  limited  value  in  clinical  diag- 
nosis. Many  prefer  to  secure  the  reaction  in  a  test-tube. 
The  dilutions  used  vary  greatly  (from  i  in  20  to  I  in  100). 
Immunized  animals  develop  the  agglutinins  in  the  blood. 
The  outlook  for  a  curative  serum  is  encouraging. 

It  now  seems  that  the  bacillus  of  Shiga  has  numerous 
close  allies,  constituting  with  it  a  "  group."  To  what  extent 
the  others  of  the  group  may  be  concerned  in  the  causation 
of  diarrhoeal  diseases,  or  may  occur  in  the  normal  intestine 
is  uncertain.  According  to  W.  H.  Park  some  of  these  form 
indol  and  develop  acid  from  mannit,  which  the  bacillus  of 
Shiga  does  not;  they  also  differ  from  it  in  their  agglutina- 
tion reactions.1 

Spirillum  cholerae  (Comma  bacillus  of  cholera). — A  rod- 
or  staff-shaped  organism,  somewhat  curved,  and  with 

1  Shiga,  Ccntralblatt  f.  Baktcriologic,  Bd.  XXIV.,  1898;  Flexner, 
Philadelphia  Medical  Journal,  September  I,  1900;  Vedder  and  Duval, 
Journal  Experimental  Medicine,  Vol.  VI.;  Gay,  University  of  Pennsyl- 
vania Medical  Bulletin.  November,  1902;  Duval  and  Bassett,  American 
Medicine,  1902,  Vol.  IV.,  p.  417;  Park  and  Carey,  Journal  Medical  Re- 
search, Vol.  IX.,  1003;  Strong  and  Mtisgrove,  Journal  American  Medical 
Association,  Vol.  XXXV.,  1900,  •).  498. 


3i6 


MANUAL    OF    BACTERIOLOGY. 


pointed  ends,  hence  the  name  "  comma "  bacillus.  The 
curved  forms,  placed  end  to  end,  may  produce  an  S-shaped 
body.  The  length  is  from  .8  to  2  n  and  the  breadth  from 


&$*!§(& 

fea>i  ^r^/rC? 
SiGS&:£.V?- 


¥ 


P.  f'*fr 

s*> 

Spirillum  of  cholera.      (X  1000.) 

.3  to  .4//.  In  cultures,  genuine  spirilla  may  be  seen.  In  the 
whitish  particles  found  in  the  stools  of  cases  of  cholera  the 
organisms  may  be  present  in  very  large  numbers.  In  these 
particles  they  may  exhibit  a  very  curious  arrangement,  lying 
parallel  with  one  another,  and,  as  remarked  by  Koch,  they 
resemble  a  school  of  fish  moving1  up  stream.  Involution 
forms,  irregular  in  outline  and  staining  poorly,  are  often 
seen  in  old  cultures.  The  organism  is  motile,  having  a 
rlagellnm  at  one  end.  It  does  not  form  spores.  It  stains 
with  the  ordinary  aniline  dyes,  but  not  by  Gram's  method. 
It  is  aerobic.  It  grows  at  the  room  temperature,  but  better 
in  the  incubator.  On  the  ordinary  media  the  growths  are 
whitish.  It  grows  best  on  neutral  or  alkaline  media,  and  is 
very  sensitive  to  a  small  amount  of  acid.  It  liquefies  gelatin. 
The  colonies  on  gelatin  plates  have  a  very  characteristic 


PATHOGENIC    BACTERIA.  317 

appearance.  They  are  nearly  round  at  first,  and  granular 
as  seen  under  the  low  power  of  the  microscope;  but  at  the 
end  of  about  twenty-four  hours  the  outline  is  slightly  irregu- 
lar, and  the  surface  looks  as  though  it  were  covered  with 
finely-broken  glass.  The  outline  later  becomes  still  more 
irregular  or  scalloped.  As  liquefaction  of  the  gelatin  takes 
place  a  funnel-shaped  depression  is  formed,  into  which  the 
colony  sinks.  The  plates  should  be  kept  at  a  temperature  of 

FIG.  91. 


Involution   forms   of  the   spirillum   of  cholera.      (Van 

from  20°  to  22°  C.  In  stab-cultures  in  gelatin  a  white 
growth  forms  around  the  stab,  and  at  the  end  of  about 
thirty-six  to  forty-eight  hours  a  funnel-shaped  depression 
occurs  at  the  surface,  owing  to  the  liquefaction  of  the 
gelatin.  This  depression  increases  in  size,  and  the  surface 
of  the  liquefied  gelatin  seems  to  be  surmounted  by  an  air- 
bubble,  which  appears  to  have  taken  the  place  of  the  part 
of  the  fluid  gelatin  which  has  evaporated.  In  the  deeper 
portion  of  the  stab  liquefaction  is  less  noticeable.  The 
growths  on  agar  are  not  characteristic.  In  bouillon  a  pellicle 
forms  on  the  surface.  On  potato  in  the  incubator  the 
growth  is  whitish  or  brownish,  not  conspicuously  elevated. 
After  growing  it  in  Dunham's  peptone  solution  in  the  incu- 


MANUAL    OF    BACTERIOLOGY. 


bator,  the  addition  of  sulphuric  acid  develops  a  red  color, 
owing  to  the  formation  of  indol  and  nitrites,  the  so-called 
"  cholera  red  "  reaction. 

The  spirillum  of  cholera  is  said  to  be  very  sensitive  to 
drying,  and,  provided  the  drying  be  complete,  is  usually 
killed  within  twenty-four  hours.  It  is  killed  in  five  minutes 
at  a  temperature  of  65°  C.  and  in  one  hour  at  55°  C.  It 

FIG.  92. 


1 


a  b  c 

Spirillum  of  cholera,  colonies  on  gelatin  plates,  X  100  to  150.      (a)  Twenty- 
four  hours  old.      (b)  Thirty  hours  old.       (c)  Forty-eight  hours  old. 
(Frankel  and  Pfeiffer.) 

may  retain  its  vitality  in  water  for  a  long  time;  observa- 
tions vary  widely  in  respect  to  determining  how  long.  In 
the  ordinary  food-substances  it  may  survive  long  enough  to 
allow  them  to  act  as  carriers  of  the  infection  if  eaten  raw. 
The  important  fact  is  that  the  cholera  spirillum  is  not  a 
strict  parasite,  but  under  favorable  conditions  it  may  main- 
tain its  vitality  for  some  time  outside  of  the  human  body. 


PATHOGENIC    BACTERIA.  319 

The  animals  ordinarily  used  for  laboratory  experiments 
are,  in  their  normal  condition,  not  susceptible  to  infection 
with  the  spirillum  of  cholera  through  the  alimentary  canal, 
and  no  animal  is  known  which  suffers  from  cholera  except- 
ing man,  though  a  disease  resembling  cholera  can  be  re- 
produced in  animals  when  certain  conditions 
are  complied  with.  In  particular  it  is 
necessary  to  avoid  the  influence  of  the  acid 
gastric  juice. 

The  following  plan  was  adopted  by 
Koch :  The  gastric  juice  was  neutralized 
with  a  solution  of  sodium  carbonate; 
the  movements  of  the  intestines  were  quieted 
by  the  injection  of  i  c.c.  of  tincture  of 
opium  for  each  200  grams  of  the  body- 
weight;  and  a  portion  of  a  pure  culture 
of  the  cholera  spirillum  was  introduced  into 
the  stomach.  When  guinea-pigs  were 
treated  in  this  manner,  in  most  cases  a 
condition  closely  simulating  cholera  was  SpirnTum  of 
produced.  The  animal  died  with  symptoms  cholera,  stab-cui- 
of  collapse.  The  small  intestine  contained  ture  in  gelatin,  two 
a  watery,  flocculent  fluid  in  which  the  spi-  d*ys  °l*  <!™k- 

*  el  and  Pfeiffer.) 

nlla  of  cholera  were  numerous.    The  mucous 
membrane  of  the  intestine  was  swollen  and  reddened. 

When  mice  or  guinea-pigs  receive  an  intra-peritoneal  in- 
jection from  a  pure  culture,  death  usually  results,  appar- 
ently from  the  toxic  substances  contained  in  the  culture. 
Pfeiffer  has  shown  that  by  repeated  doses,  insufficient  to 
kill  the  animal,  of  cultures  whose  vitality  has  been  de- 
stroyed by  heat  or  otherwise,  the  animal  may  be  made  im- 
mune. He  has  also  shown  that  when  living  comma  bacilli 
are  introduced  in  the  peritoneum  of  an  immune  animal  they 
are  rapidly  destroyed  and  disintegrated  (see  page  189).  He 


32O  MANUAL    OF    BACTERIOLOGY. 

has  advised  the  use  of  this  reaction  as  a  means  of  diagnosis, 
inasmuch  as  the  spirilla  which  apparently  resemble  the 
spirillum  of  cholera,  but  are  in  reality  different  from  it,  do 
not  become  disintegrated  when  they  are  introduced  in  the 
peritoneum  of  an  animal  made  immune  to  the  spirillum  of 
cholera.  It  has  been  shown  also  that  blood  from  animals 
made  immune  to  cholera  has  an  agglutinating  action  upon 
the  spirillum  of  cholera  like  that  seen  when  the  blood-serum 
of  cases  of  typhoid  fever  is  mixed  with  living  typhoid 
bacilli. 

The  outlook  is  encouraging  for  the  production  of  a  safe  method  for 
immunizing    healthy    persons    from    cholera    during    an    epidemic.1 

Although  a  positive  demonstration  that  the  spirillum  of 
Koch  is  the  cause  of  cholera  is  lacking,  as  far  as  the  exact 
reproduction  of  the  disease  in  animals  is  concerned,  the 
necessary  proof  has  been  supplied  by  the  accidental  or 
intentional  infection  of  laboratory  investigators  who  were 
working  with  cholera,  which  has  happened  on  several  occa- 
sions. 

Bacteriological  investigations  of  the  victims  of  cholera 
have  shown  that  the  spirilla  of  cholera  are  present  in  very 
large  numbers  in  the  watery  contents  of  the  intestine,  espe- 
cially early  in  the  disease.  They  appear  in  the  lumina  of 
the  glands,  and  they  may  be  seen  underneath  the  epithelial 
cells.  They  may  occur  in  the  matters  vomited.  They 
usually  are  not  found  widely  spread  through  the  organs  of 
the  body.  It  is  probable  that  the  symptoms  of  the  disease 
result  from  poisonous  substances  produced  by  the  spirilla 
or  contained  in  them. 

The  infectious  element  in  cholera  is  usually  transmitted 
through  water,  and  numerous  epidemics  have  been  studied 
where  the  infection  was  traced  to  drinking-water,  and  the 
origin  of  the  contamination  was  discovered.  The  organ- 

1  Strong,  American  Medicine,  Aug.  15,  19x53. 


f 

.     PATHOGENIC    BACTERIA.  321 

isms  may,  however,  be  carried  by  other  articles  of  food, 
and  may  be  conveyed  occasionally  through  contaminated 
clothing  and  bedding,  and  probably  by  flies.  The  excreta 
and  bedding  should  be  thoroughly  sterilized,  the  hands  of 
the  attendants  must  be  carefully  disinfected.  Although 
commoner  in  the  summer-time,  epidemics  of  cholera  have 
been  known  to  occur  in  the  winter. 

Bacteriological  Diagnosis  of  Cholera. — When  cases  sus- 
pected of  being  cholera  appear  in  a  community,  it  becomes 
a  matter  of  the  utmost  importance  to  determine  the  exact 
nature  of  the  disease  in  order  that  it  may  not  become 
epidemic.  One  of  the  first  occasions  when  bacteriological 
methods  were  put  into  practice  in  the  diagnosis  of  cholera 
was  at  the  time  of  the  appearance  of  that  disease  in  the 
Port  of  New  York  in  1887. 

According  to  Koch,  the  diagnosis  may  be  made  in  twenty- 
four  hours  or  less.  It  is  important  to  obtain  the  discharges 
from  the  intestines  as  early  in  the  course  of  the  disease  as 
possible,  and  while  they  are  perfectly  fresh.  It  may  be 
necessary,  however,  to  examine  the  moist  dejecta  on  the 
linen  or  clothing,  when  no  other  material  is  available. 

In  the  first  place,  one  of  the  small,  partly-solid  particles 
which  may  be  found  in  the  discharges  from  the  intestines 
should  be  smeared  upon  a  cover-glass,  fixed  in  the  usual 
manner,  stained  with  one  of  the  aniline  dyes,  and  examined 
with  the  microscope.  If  taken  early  in  the  disease,  the 
comma  bacilli  may  be  present  in  large  numbers,  and  they 
are  likely  to  be  arranged  in  more  or  less  parallel  groups 
(see  above).  If  comma-shaped  bacilli  are  thus  found,  a 
strong  probability  is  created  that  the  disease  is  Asiatic 
cholera.  The  motility  of  the  organisms  can  be  determined 
by  examination  in  the  hanging-drop.  It  is  to  be  remem- 
bered that  spirilla  of  various  forms  are  common  in  the 
normal  mouth,  and  may  appear  in  the  stools  (see  pages 
152  and  228). 


322  MANUAL    OF    BACTERIOLOGY. 

The  diagnosis  should  be  confirmed  by  the  use  of  culture- 
methods.  Using  the  small,  semi-solid  particles  from  the 
intestinal  discharges,  gelatin  plates  in  the  usual  three  dilu- 
tions (see  page  97)  should  be  made  and  kept  at  a  tempera- 
ture of  20°  to  22°  C.  At  the  end  of  twenty-four  hours  or 
less  the  colonies  of  the  spirillum  of  cholera  should  have 
been  developed  and  should  present  the  picture  characteristic 
for  these  colonies  in  gelatin  plates  (Fig.  92),  which  enables 
them  to  be  differentiated  from  colonies  of  other  bacteria. 
From  one  of  these  colonies,  preparations  may  be  made  for 
microscopic  examination,  and  a  set  of  tubes  may  be  inocu- 
lated. The  most  characteristic  growth  will  be  from  stick- 
cultures  in  gelatin.  The  growth  in  Dunham's  peptone 
solution  may  be  tested  for  the  development  of  indol  and 
nitrites. 

At  the  time  that  the  first  smear  preparations  and  gelatin 
plates  are  prepared,  tubes  of  peptone  solution  should  be 
inoculated  directly  from  the  intestinal  contents,  and  kept 
in  the  incubator  (Schottelius).  After  development  has  oc- 
curred, the  production  of  indol  may  be  tested  by  the  addi- 
tion of  sulphuric  acid.  These  tubes  are  especially  valuable 
when  unfavorable  material  or  when  material  containing 
small  numbers  of  the  spirilla  is  used.  In  the  incubator  the 
spirilla  may  be  expected  to  multiply  in  the  peptone  solution 
rapidly,  and  to  appear  upon  the  surface  of  the  liquid  in 
large  numbers,  even  forming  a  visible  film  in  six  hours. 
Smears  may  be  made  from  the  surface  part  of  these  tubes, 
stained,  and  examined  with  a  microscope.  From  the  same 
material  gelatin  plates  should  be  prepared,  and  examined 
as  soon  as  the  colonies  develop. 

\Yhen  cultures  are  obtained,  their  effects  may  be  tested 
upon  guinea-pigs  by  injecting  them  into  the  peritoneum. 
The  reaction  described  by  Pfeiffer  as  resulting  from  the 
injection  of  cholera  spirilla  into  the  peritoneum  of  immune 


PATHOGENIC    BACTERIA.  323 

animals  has  been  recommended  as  an  additional  means  of 
diagnosis  between  the  cholera  spirillum  and  related  forms. 
The  agglutinating  power  which  the  blood  of  animals  im- 
munized to  cholera  has  for  the  cholera  spirillum  may  be 
employed  in  the  same  way. 

In  the  examination  of  water  for  the  spirillum  of  cholera, 
to  i  liter,  or  more,  of  water,  add  enough  of  a  strong  pep- 
tone solution  to  make  it  contain  i  per  cent,  peptone  and 
.5  per  cent,  sodium  chloride.  (The  strong  peptone  solution 
contains  20  per  cent,  peptone  and  10  per  cent,  sodium 
chloride;  is  alkaline  and  sterile.)  The  water,  with  the 
peptone  in  it,  is  divided  among  a  number  of  sterilized 
flasks.  After  twelve  hours  in  the  incubator,  any  vibrios  in 
it  are  likely  to  have  multiplied  and  to  have  formed  a  scum 
on  the  surface,  which  may  be  investigated  for  the  char- 
acteristics of  the  spirillum  of  cholera  according  to  the 
methods  given  above.  See  also  page  142. 

Since  Koch's  discovery  of  the  cholera  spirillum  in 
1883-84  a  considerable  number  of  bacteria  have  been 
described  which  resemble  the  cholera  spirillum  more  or 
less  closely,  and  which  have  to  be  taken  into  account  in 
making  examinations  of  material  of  any  sort  for  it.  This 
is  particularly  necessary  in  the  investigation  of  water,  in 
which  such  spirilla  seem  to  occur  quite  frequently. 

Vibrio  Metchnikovi.  —  A  comma-shaped  organism, 
which  though  somewhat  shorter  and  thicker  may  be  very 
similar  to  the  comma  bacillus  of  cholera  in  form,  and  which, 
like  it,  may  sometimes  form  genuine  spirilla.  It  is  motile 
and  has  a  flagellum  at  one  end.  It  does  not  form  spores. 
It  is  aerobic.  It  stains  with  the  aniline  dyes,  and  is  not 
stained  by  Gram's  method.  It  grows  at  the  room  tempera- 
ture. It  liquefies  gelatin  somewhat  more  rapidly  than  the 
spirillum  of  cholera.  The  colonies  on  gelatin  plates  are 
not  all  alike;  some  of  them  resemble  those  of  vibrio  pro- 
tens,  and  others  are  extremely  like  those  of  the  spirillum  of 
28 


324  MANUAL    OF    BACTERIOLOGY. 

cholera.  It  grows  upon  the  usual  media.  Blood-serum  is 
liquefied  by  it.  The  growth  on  agar  is  grayish  to  yellowish, 
and  abundant.  It  forms  a  pellicle  on  bouillon.  In  milk 
an  acid  reaction  is  developed  with  coagulation.  In  peptone 
solution  it  produces  indol  and  nitrites  like  the  spirillum  of 
cholera.  It  is  said  to  lead  to  the  production  of  indol  more 
intensely  than  the  spirillum  of  cholera. 

It  is  killed  by  a  temperature  of  50°  C.  in  five  minutes. 
It  was  discovered  in  chickens  suffering  from  gastro-ente- 

FIG.  94. 


ritis.  It  is  pathogenic  to  chickens,  pigeons  and  guinea- 
pigs,  less  so  to  mice  and  to  rabbits.  The  comma-shaped 
organisms  are  found  in  the  blood  in  guinea-pigs,  pigeons 
and  young  chickens. 

Vibrio  proteus  (of  Finkler  and  Prior). — A  comma-shaped 
organism  somewhat  larger  than  the  spirillum  of  cholera, 
sometimes  exhibiting  genuine  spiral  forms,  and  also,  at 

1The  magnification   is  a  little  greater  than  in  the  other  photomicro- 
graphs. 


PATHOGENIC    BACTERIA.  325 

times,  involution  forms.  It  is  motile  and  has  a  flagellum 
at  one  end.  It  liquefies  gelatin  much  more  rapidly  than 
the  spirillum  of  cholera,  and  the  colonies  in  gelatin  develop 
more  rapidly.  At  the  end  of  twenty-four  hours  the  colo- 
nies are  uniformly  circular,  larger  than  those  of  the  spiril- 
lum of  cholera,  and  uniformly  granular,  when  slightly 
magnified.  On  the  other  culture-media  the  growths  are 
usually  whitish.  On  potato  it  produces  an  abundant,  moist, 
grayish-yellow  deposit,  and  grows  at  the  room  temperature. 
It  liquefies  blood-serum;  milk  becomes  acid.  In  peptone 
solution  it  does  not  form  indol.  It  is  less  pathogenic  to 
animals  than  the  spirillum  of  cholera.  It  was  supposed  by 
its  discoverers  to  be  the  cause  of  cholera  nostras,  but  it  ap- 
pears to  have  no  relation  to  that  disease. 

Spirillum  Milleri. — A  comma-shaped  organism  resem- 
bling vibrio  proteus  in  many  respects,  and  probably  ident- 
ical with  it.  In  gelatin  it  grows  more  rapidly,  and  produces 
liquefaction  more  rapidly  than  the  spirillum  of  cholera. 
On  gelatin  plates,  at  the  end  of  twenty-four  hours,  the 
colonies  are  uniformly  circular  and  granular,  lying  in  little 
depressions  resulting  from  the  liquefaction  of  the  gelatin. 
Its  growths  in  the  other  media  are  not  characteristic.  It 
liquefies  blood-serum.  It  does  not  produce  indol.  It  is 
less  toxic  to  animals  than  the  spirillum  of  cholera.  It  was 
isolated  by  Miller  from  a  carious  tooth. 

See  also  Spirillum  sputigenum,  Part  III. 

Spirillum  tyrogenum  (of  Deneke). — A  comma-shaped 
organism  not  so  large  as  the  spirillum  of  cholera.  It  is 
motile,  having  a  flagellum  at  one  end.  It  does  not  form 
spores.  In  cultures,  genuine  spirilla  may  develop.  Gelatin 
is  liquefied  more  rapidly  than  by  the  spirillum  of  cholera, 
and  the  colonies  develop  more  rapidly.  The  circumference 
of  the  colony  is  round,  the  surface  may  appear  somewhat 
granular,  and  it  has  a  greenish-brown  color,  seen  under 


326  MANUAL    OF    BACTERIOLOGY. 

the  low  power.  The  colonies  differ  noticeably  from  the 
colonies  of  the  cholera  spirillum,  in  the  more  rapid  lique- 
faction of  gelatin.  Milk  containing  litmus  becomes  acid, 
is  subsequently  decolorized,  and  is  also  coagulated.  It 
liquefies  blood-serum.  It  does  not  form  indol  in  Dunham's 
peptone  solution.  No  pellicle  forms  in  cultures  upon  bouil- 
lon. It  is  less  toxic  to  animals  than  the  spirillum  of  cholera. 
It  was  isolated  originally  from  old  cheese. 

Vibrio  Berolinensis. — A  comma-shaped  organism  resem- 
bling the  spirillum  of  cholera  in  form  and  in  the  position 
of  its  flagellum.  It  does  not  stain  by  Gram's  method.  It 
grows  at  the  room  temperature,  but  more  rapidly  in  the 
incubator.  The  colonies  upon  gelatin,  one  or  two  days  old, 
when  magnified,  are  decidedly  more  finely  granular  and 
more  transparent  than  those  of  the  spirillum  of  cholera,  and 
the  margin  is  almost  absolutely  smooth  and  circular.  As 
the  colonies  become  older  they  assume  a  more  irregular 
and  lobulated  appearance,  but  are  still  more  finely  granular 
than  the  colonies  of  the  cholera  spirillum.  Gelatin  is  very 
slowly  liquefied.  Its  growth  on  the  other  culture-media  is 
not  remarkable.  It  forms  indol  in  peptone  solution,  and  it 
increases  in  the  upper  layers  of  the  fluid.  When  guinea- 
pigs  are  inoculated  in  the  peritoneal  cavity,  death  occurs  in 
one  to  two  days.  This  organism  was  discovered  in  the 
water-supply  of  Berlin. 

Other  spirilla  have  been  isolated  from  water  by  Gunther 
(vibrio  aquatilis  in  Spree  water)  ;  by  Dunbar  from  the  Elbe 
River;  by  Russell  from  the  Gulf  of  Naples;  by  Heider 
from  the  water  of  the  Danube  Canal ;  and  in  America,  by 
Abbott,  from  the  water  of  the  Schuylkill  (vibrio  Schuyl- 
kiliensis)  ;  and  many  others  have  been  described  to  which 
the  limits  of  this  work  will  not  permit  of  further  allusion. 

The  Spirillum  or  Spirochaeta  Obermeieri  (of  relapsing 
fever). — A  slim  spirillum  with  numerous  turns,  16  to  40 /* 


PATHOGENIC    BACTERIA.  327 

in  length.  The  ends  are  pointed.  It  is  actively  motile. 
The  spirillum  is  not  stained  by  Gram's  method  but  may  be 
colored  by  the  ordinary  aniline  dyes.  The  organism  has 
never  been  cultivated.  It  is  found  abundantly  in  the  blood 

FIG.  95. 


Spirillum  of  Relapsing  Fever  in  the  Blood.     Sketched  from  a  Stained 

Specimen. 

and  in  the  spleen  during  the  attack  of  fever.  The  spleen 
is  enlarged.  The  disease  has  been  produced  in  apes  by 
inoculating  them  with  blood  taken  from  men  having  the 
disease. 

It  is  asserted  that  the  spirillum  is  transferred  by  bed- 
bugs from  one  person  to  another.1 

1  Karlinski,   Centralblatt  f.   Baktcriologie,  Bd.   XXXI.,   Orig.,    1902. 


328  MANUAL    OF    BACTERIOLOGY. 


APPENDIX. 
PATHOGENIC    PROTOZOA. 

PROTOZOA  are  unicellular  animal  organisms.  As  they  are 
studied  by  methods  that  have  much  in  common  with  those 
used  for  the  bacteria  they  may  be  considered  here  briefly. 
Protozoa  are  numerous  in  pond  and  ditch  water,  and  these 
species  seem  to  be  harmless.  However,  many  diseases  of 
the  lower  animals  are  caused  by  protozoa,  such  as  surra, 
Texas  fever,  and  coccidium  disease  of  rabbits.  Birds,1  rep- 
tiles and  frogs2  may  show  organisms  in  the  blood,  resem- 
bling the  parasites  of  malaria.  Until  recently  it  has  been 
doubtful  whether  any  pathogenic  protozoon  has  ever  been 
propagated  in  pure  culture  outside  of  the  body  of  the  host. 
This  has  recently  been  accomplished  by  Novy  and  McXcal 
for  a  parasite  (Trypanosoma)  from  the  blood  of  the  rat 
on  rabbit-blood-agar. 

Amoeba  Dysenterise  (Amoeba  Coli). — Associated  with 
annt'bic  dysentery  and  believed  to  be  its  causative  agent  is 
the  (juurba  d \scntcr ice,  more  often  named  amcrba  coli. 
These  organisms  are  found  in  the  intestinal  ulcers,  the  feces, 
the  secondary  liver  abscesses  and  the  sputum  (in  the  latter 
only  when  an  amoebic  liver  abscess  has  perforated  into  the 
lung).  The  lesion  in  the  colon  is  a  severe  inflammation 
accompanied  by  necrosis  chiefly  of  the  submucous  layer,  and 
leading  to  extensive  ulceration.3  According  to  Strong,  at 
least  two  distinct  species  of  amoebae  have  been  found  in  the 

1  Opie.   MacCallum,  Journal  Experimental  Medicine,  Vol.  III. 
•"Lnnirniann.  AYu'  Y<  rk  Medical  Journal,  January  7,  1899. 
3  See  Councilman  and  Laflcur.  Johns  Hopkins  Hospital  Reports,  Vol. 
II.;  soo  also  Harris,  American  Journal  Medical  Sciences,  Vol.  115,  1898. 


APPENDIX.  329 

feces  in  man,  only  one  of  which  is  pathogenic  and  the  cause 
of  dysentery.  Unfortunately  the  designation,  amoeba  coli, 
has  been  applied  to  both  species.  The  amoeba  of  dysentery 
should  be  designated  amoeba  dysenteries,  limiting  the  term 
amoeba  coli  to  the  non-pathogenic  form  or  forms. 

The  amoeba  dysenteric  is  a  unicellular  organism,  20-50  /* 
in  diameter  when  at  rest,  consisting  of  a  clear,  homogeneous 
ectosarc  and  a  granular  endosarc,  with  an  eccentrically 
placed  nucleus.  The  endosarc  contains  a  number  of  vacu- 
oles  of  variable  size  and  very  frequently  red  blood-cor- 
puscles, as  well  as  other  foreign  bodies  such  as  bacteria, 
pigment  granules,  etc.  Many  red  blood-corpuscles  may  be 
seen  crowded  together  in  a  single  amoeba.  The  organism  is 
actively  amoeboid,  extending  its  substance  into  processes 
or  pseudopodia  of  varying  forms.  This  amoeboid  motion 
assists  in  making  easy  the  recognition  of  the  parasites  under 
the  microscope  and  in  distinguishing  them  from  large, 
swollen  cells  found  in  the  feces.  The  stool  should  be  ex- 
amined while  fresh  and  still  warm. 

The  non-pathogenic  amoeba  (amoeba  coli),  also  occasion- 
ally found  in  the  intestinal  tract  of  man,  differs  from  the 
pathogenic  dysenteric  organism  chiefly  in  its  much  smaller 
size  ( 10-24  /;)  and  the  invariable  absence  of  red  corpuscles 
from  its  interior.  The  protoplasmic  granules  are  also,  as  a 
rule,  smaller  and  are  difficult  to  recognize.  The  amoeba 
dysenteric  produces  experimentally  definite  ulceration  of 
the  gut  of  cats,  whereas  the  amoeba  coli  is  harmless.  Both 
varieties  of  amoebae  may  be  stained  by  a  special  stain  de- 
vised by  Mallory.2 

1  Strong,    Circulars    on    Tropical    Diseases,    No.    I,    Chief    Surgeon's 
Office,   Headquarters,   Division  of  the   Philippines,   Manila,   P.   I.,  Feb- 
ruary, 1901.    Ibid.,  No.  IT,  April,  1901.     (Both  reports  may  be  obtained 
from  the  United  States   Government,  Washington.) 

2  Mallory,  Journal  of  Experimental  Medicine,   September,   1897,   Vol. 
II.,  p.  529. 


33°  MANUAL    OF    BACTERIOLOGY. 

The  Malarial  Parasite.1  (Plasmodium  or  Hematozoon 
Malaria). — The  organisms  of  malaria  consist  of  at  least 
three  different  species,  each  associated  with  one  of  the  three 
types  of  malarial  fever:  The  tertian  parasite  with  benign 
tertian  malarial  fever,  the  parasite  reaching  maturity  in 
forty-eight  hours;  the  quartan  parasite  with  benign  quar- 
tan malarial  fever,  the  cycle  of  development  requiring 
seventy-two  hours;  and  the  &stivo-autumnal  parasite  with 
malignant,  aestivo-autumnal  fever,  developing  to  maturity 
in  a  variable  period  of  from  twenty-four  to  forty-eight 
hours.  The  parasites  are  studied  to  best  advantage  in  a 
drop  of  fresh,  fluid  blood  placed  between  a  cover-glass  and 
slide  and  examined  with  an  oil-immersion  objective.  For 
method  of  making  and  staining  dry  preparations,  see  pages 
55  and  108. 

Tertian  Parasite. — This  appears  in  its  youngest  form  as 
a  small,  round,  colorless,  hyaline  body  within  the  red  cor- 
puscle, seen  during  and  just  after  the  chill  of  the  disease. 
This  body  may  be  actively  amu'boid.  suddenly  changing  its 
contour  into  various  forms.  Its  size  gradually  increases, 
and  fine,  dark,  actively-motile,  dancing  pigment  granules 
begin  to  appear  at  its  periphery. 

The  red  corpuscle  harboring  the  parasite,  with  the  growth 
of  the  latter,  becomes  gradually  paler  and  expands  in  size. 
The  parasite  as  it  grows  loses  its  earlier  amoeboid  movement 
and  the  pigment  granules,  still  actively  motile,  accumulate. 
Xear  the  end  of  forty-eight  hours,  the  organism  finally  fills 
the  red  corpuscle,  only  a  faint  rim  indicating  the  latter.  The 
ripe  parasite  now  divides  it  into  from  fifteen  to  twenty-live 
small,  round,  hyaline  spores  which  are  arranged  somewhat 
radially  about  the  pigment  granules  which  have  lost  their 
motility  and  become  concentrated  in  a  clump  at  the  center  of 

1  Thaver  and  llewetson,  "The  Malarial  Fevers  of  Kaltimore."  Johns 
Hopkins  Hospital  Reports,  iS<>5.  Vol.  V.,  and  reprinted;  Thayer,  "Lec- 
tures on  the  Alalarial  Fevers,"  Xe\v  York, 


APPENDIX. 


331 


the  spore- forming  organism.  The  spores  finally  break  apart 
and  scatter,  each  destined  to  invade  a  red  corpuscle  and 
start  anew  the  cycle  of  development.  This  cycle  may  be 
repeated  over  and  over  again,  producing  a  corresponding 
number  of  malarial  paroxysms. 

FIGS.  96-99. 


o 


Malarial  Parasites  in  Various  Stages.  (X  1000.)  96,  97  and  98  are 
tertian  parasites  ;  98  shows  the  completion  of  segmentation.  99  is  the 
crescentic  form  of  the  festivo-autumnal  parasite. 

Certain  full-grown  parasites  do  not  complete  the  cycle  of  development 
by  sportilation,  as  described,  but,  breaking  loose  from  the  corpuscle, 
remain  as  "extra-cellular"  bodies.  These  are  seen  chiefly  after  the 
paroxysm  as  large,  round,  pale  bodies  containing  numerous  dancing 
pigment  granules  scattered  through  their  substance.  They  ultimately 
degenerate  and  disappear.  Some  of  these  extra-cellular  forms  may  be 
seen  to  develop  long  slender  processes  (flagella),  having  a  very  active 
whip-like  motion.  Flagella  are  never  observed  in  perfectly  fresh  blood 
but  develop  only  after  the  blood  has  been  drawn  some  time,  usually 
fifteen  or  twenty  minutes. 


332  MANUAL    OF    BACTERIOLOGY. 

The  extra-cellular  forms  of  the  parasite  (gametes),  incapable  of  fur- 
ther development  in  their  human  intermediate  host,  can  continue  their 
life  cycle  only  when,  by  chance,  they  happen  to  be  sucked  into  the  body  of 
a  mosquito  of  the  genus  Anopheles  (definite  host),  in  which  they  un- 
dergo a  second  complete  (sexual)  cycle  of  development  with  the  ultimate 
production  of  spores  (sporozoids).  When  in  turn  the  spores  chance  to 
be  inoculated  into  the  blood  of  man  by  the  bite  of  an  infected  Anopheles, 
the  man  becomes  infected,  and  the  cycle  of  development  in  the  red 
corpuscle,  already  outlined,  commences.  The  second  or  sexual  cycle  of 
the  parasite  in  the  mosquito,  here  described  for  the  tertian  organism, 
applies  as  well  to  the  other  varieties  of  the  malarial  organism,  namely 
the  quartan  and  the  aestivo-autumnal  forms,  in  the  case  of  each  starting 
from  the  extra-cellular  mature  forms  of  the  organism  found  in  the 
blood  of  the  human  host.1 

Quartan  Parasite. — This  resembles  quite  closely  the  ter- 
tian parasite,  but  differs  from  it  in  certain  respects.  The 
young,  hyaline,  intra-corpuscular  parasite  is  more  highly 
refractive,  its  amoeboid  motion  is  less  marked  and  more 
sluggish,  and  the  pigment  granules  are  darker,  much  coarser, 
and  have  very  slight  motility.  The  infected  red  corpuscles 
are  usually  somewhat  contracted  instead  of  swollen  and 
their  color  is  apt  to  be  darker,  assuming  a  bronzed  hue.  The 
full-grown  parasite  is  much  smaller  than  the  corresponding 
form  of  the  tertian,  approximating  the  size  of  a  normal  red 
corpuscle.  As  segmentation  begins,  a  characteristic  appear- 
ance develops  which  distinguishes  the  quartan  organism, 
namely,  the  coarse  pigment  granules  are  drawn  toward  the 
center  of  the  parasite  in  certain  converging  straight  paths, 
giving  a  stellate  arrangement  to  the  pigment,  until  finally  it 
becomes  clumped  entirely  at  the  center  in  a  solid  mass.  The 
segmenting  forms  of  the  quartan  parasite  thus  present  a 
more  symmetrical  arrangement  of  the  spores,  which  often 
resemble  the  petals  of  a  "  marguerite."  These  spores  are 
oval  and  number  only  from  six  to  twelve,  being  fewer  than 
those  of  the  tertian  segmenting  parasite.  The  quartan 

1  Lyon,  "The  Inoculation  of  Malaria  by  the  Mosquito:  A  Review  of 
the  Literature,"  Medical  Record,  February  17,  1900. 


APPENDIX.  333 

extra-cellular  forms  are  smaller  than  those  of  the  tertian, 
being  about  the  size  of  a  red  corpuscle,  and  contain  coarse 
pigment  granules  in  active  motility  until  degeneration 
occurs.  Flagella  may  develop  from  certain  extra-cellular 
forms.  The  entire  development  of  the  quartan  parasite 
occupies  about  seventy-two  hours. 

/Estwo-autumnal  Parasite. — This  parasite  develops  to 
maturity  in  from  twenty-four  to  forty-eight  hours  and  is 
usually  regarded  as  representing  a  single  species,  though 
certain  observers  claim  to  distinguish  two  distinct  varieties. 
The  usual  description  of  a  single  variety  is  here  adopted. 
The  youngest  forms  (hyaline  bodies)  resemble  those  of  the 
tertian  and  quartan  organisms,  but  are  distinctly  smaller  and 
more  highly  refractive.  They  often  present  a  ring-like 
appearance.  They  are  amoeboid.  Pigment  granules  later 
appear  at  their  periphery,  but  are  exceedingly  minute  and 
scanty,  seldom  more  than  one  or  two  being  seen.  These 
granules  have  little  or  no  motility  and  in  fact  are  with  diffi- 
culty made  out.  The  hyaline  bodies  remain  small,  seldom 
exceeding  one  third  the  diameter  of  a  red  corpuscle.  The 
infected  corpuscle  is  apt  to  be  crenated,  shrunken  and  dark. 
These  are  the  forms  seen  in  the  circulating  blood  in  early 
infections;  the  mature  forms,  with  the  exception  of  the 
extra-cellular  forms,  developing  in  the  spleen  and  bone- 
marrow,  rarely  reach  the  general  circulation.  Blood  from 
the  spleen  shows  the  full-grown  forms  in  abundance.  The 
segmenting  forms  resemble  those  of  the  tertian  parasite 
both  in  the  numbers  of  the  segments  and  in  their  arrange- 
ment, but  are  much  smaller  in  the  aggregate,  as  well  as  in 
the  individual  segments. 

After  the  fever  has  lasted  about  one  week,  extra-cellular 
forms  make  their  appearance  in  the  circulating  blood.  These 
are  crescentic,  ovoid  or  small  round  bodies,  containing  coarse 
pigment  granules  at  their  center,  generally  arranged  in  a 


334  MANUAL    OF    BACTERIOLOGY. 

ring.  The  crescents  and  ovoid  bodies  are  highly  refractive 
and  are  in  length  about  equal  to  the  diameter  of  a  red  cor- 
puscle, sometimes  larger.  The  round  forms  are  smaller  than 
a  red  corpuscle,  with  the  pigment  arranged  centrally  in  a 
ring.  They  may  become  flagellated  after  the  blood  has 
remained  outside  the  body  for  some  minutes.  Any  of  the 
extra-cellular  bodies  may  show  remnants  of  the  red  cor- 
puscle attached  to  its  side,  like  a  bib.  The  extra-cellular 
forms  are  concerned  in  the  cycle  of  development  of  the 
organism  in  the  mosquito,  and  are  sterile  in  the  human 
body.  They  are  exceedingly  resistant  to  quinine  and  may 
continue  in  the  blood  for  long  periods  of  time. 

Melaniferous  leucocytes  (phagocytes)  are  seen  in  the 
blood,  being  especially  abundant  after  the  paroxysm  in  all 
forms  of  malarial  infection.1 

Small-pox  and  Vaccinia. — Micrococci  of  various  sorts 
have  been  found  in  the  pustules  of  small-pox  and  vaccinia, 
but  indicate  only  a  secondary  infection.  Other  microorgan- 
isms have  been  described.  The  most  important  are  certain 
bodies  often  considered  protozoa.  In  both  small-pox  and 
vaccinia  small  round  homogeneous  bodies,  2  to  4  /*  in 
diameter,  have  been  found  in  the  epithelial  cells  of  the 
vesicles.  Inoculation  of  vaccine  lymph  into  the  rabbit's 
cornea  leads  to  the  production  of  similar  bodies  in  the 
epithelial  cells  of  the  cornea.  W.  Reed2  found  small  amoe- 
boid bodies  in  the  blood  in  cases  of  small-pox  and  vaccinia. 
Vaccine  virus  that  has  been  filtered  through  the  Chamber- 
land  or  Berkenfeld  filter  is  no  longer  active.  From  this  it 
may  be  presumed  that  the  organism  causing  it  is  not  too 
small  to  be  seen  with  the  microscope. 

Councilman,   Magratli  and   Brickerhoff,3   as  a  result  of 

'See  also  E\vin.u\  Journal  Experimental  Medicine,  Yols.   V.  and  VI. 

'Journal  Experimental  Medicine,  Vol.  11..  515;  sec  also,  Anna  Wil- 
liams and  Flournoy,  and  W.  H.  Park,  N.  Y.  Univ.  Bull  Medical 
Sciences,  Tl.,  October,  1902. 

3  Journal  Medical  Research.  Vol.  IX.,  May,  1903. 


APPENDIX.  335 


recent  studies,  believe  that  the  bodies  above  mentioned  are 
protozoa.  Segmentation  of  the  bodies  is  described,  result- 
ing in  the  formation  of  spore-like  bodies.  The  spore-like 
bodies  undergo  a  further  or  second  cycle  of  development 
within  the  nucleus.  The  second  cycle  also  ends  in  segmenta- 
tion. The  two  cycles  were  seen  in  small-pox;  in  vaccinia, 
only  the  first  or  extranuclear  bodies  were  observed. 

YELLOW    FEVER. 

It  has  already  been  indicated  (page  160)  that  the  study  of  cases  of 
yellow  fever  has  failed  to  prove  that  this  disease  is  caused  by  bacteria. 
On  the  other  hand,  evidence  that  it  is  transmitted  by  the  mosquito, 
Stegomyia,  has  been  increasing. 

As  malaria  and  some  other  diseases  transmitted  by  mosquitoes  are  due 
to  protozoa,  careful  search  has  been  made  for  protozoa  in  yellow  fever. 
Examinations  of  the  blood  of  individuals  having  yellow  fever  have  been 
without  result.  Recently  Parker,  Beyer  and  Pothier  have  studied  speci- 
mens of  Stegomyia  fed  on  such  blood.  They  claim  to  have  found  that 
these  mosquitoes  became  infected  with  a  protozoon  parasite,  a  portion 
of  whose  cycle  was  worked  out.1  Ordinary  mosquitoes  did  not  contain 
the  parasite.  Blood-serum  from  a  case  of  yellow  fever  was  filtered 
through  a  Berkenfeld  filter  and  injected  into  two  healthy  subjects;  one 
subject  developed  yellow  fever  and  the  other  did  not.  The  interpreta- 
tion of  these  results  in  connection  \vith  the  experiments  of  Reed  and 
Carroll  (page  161)  is  not  at  present  clear. 

Trypanosomes.— A  number  of  species  of  Trypanosoma  have  been  de- 
scribed, which  produce  diseases  in  the  lower  animals;  recently  one  has 
been  stated  to  be  the  cause  of  disease  in  man.2  The  trypanosoma  is  a 
protozoon  belonging  to  the  flagellata.  It  is  of  an  elongated  spindle- 
shaped  form,  with  a  nucleus,  and  has  a  flagellum  at  one  end,  which 
extends  along  a  thin  edge,  called  the  undulating  membrane.  It  is  ac- 
tively motile.  It  occurs  in  the  blood,  between,  but  not  in,  the  blood- 
corpuscles.  Its  length  is  two  to  several  times  the  diameter  of  a  red 
corpuscle.  Members  of  this  genus  are  the  cause  of  surra  (a  fatal  dis- 

1  Marine  Hospital  Service.     Yellow  Fever  Institute,  Bulletin  No.  13, 
March,  1903. 

2  For  a  full  description  of  the  life  history  and  classification  of  Trypan- 
osoma   see    Salmon   and    Stiles,    Emergency   Report   on    Surra.     U.    S. 
Bureau    Animal    Industry,    Bulletin    No.    42,    1902.      See    also    Francis, 
Marine  Hospital  Service,  Hygienic  Laboratory,  Bulletin  No.   n,   1903. 


336  MANUAL   OF    BACTERIOLOGY. 

ease  of  horses  and  mules  occurring  in  India  and  the  Philippine  islands) 
and  of  the  tsetse-fly  disease  of  South  Africa;  while  others  are  found 
in  rats,  birds,  amphibia  and  fishes.  In  the  horse  the  infection  is  trans- 
mitted by  the  bites  of  flies.  Novy  and  McNeil  have  succeeded  in  culti- 
vating the  trypanosoma  of  rats  on  rabbit  blood-agar.1 

Several  cases  were  reported  during  1902  where  trypanosomes  were 
found  in  the  b  ood  of  individuals  from  tropical  Africa,  showing  that 
this  group  of  parasites  may  occur  in  man.2  The  symptomatology  of 
these  infections  requires  further  study.  Still  more  recently  it  has  been 
claimed  by  Castellani  that  a  trypanosoma  is  the  cause  of  "  sleeping  sick- 
ness," a  disease  of  the  natives  of  Africa.  He  states  that  the  parasites 
may  be  demonstrated  in  the  cerebro-spinal  fluid  obtained  by  lumbar 
puncture  and,  with  greater  difficulty,  in  the  blood,  during  life.  Many 
cases  also  show  at  autopsy  streptococcus  infection,  which  is  believed  to 
be  a  secondary  invasion.3 

1  Novy  and  McNeil,  in  Contributions  to  Medical  Research  dedicated 
to  Victor  C.  Vaughan,  1903. 

2  British  Medical  Journal,  May  30,  1903. 

3Britisli  Medical  Journal  and  Lancet,  June  20,  1903. 


Surface  Divided  in  Square  Centimeters  for  Counting  Colonies, 


O/  6 

Plate  for  Counting  Colonies  of  Bacteria  in  Petri  Dishes. 


INDEX. 


ABBE  condenser,  31 
Abrin,    174,    181 
Abscesses,    235,   245 

metastatic,    242 
Absorbent  cotton,  82,  92,  219 
Acetic  acid,  40,   52,    130 
Accidental    infection    of    laboratory 

workers,    115 
Acid,    acetic,   40,    52,    130 

alcohol,  42,  45 

aniline   dyes,  39 

boric,    207 

butyric,  130,  224 

carbolic,    200,    129,    197,    220 

formic,   130 

fuchsin,    39 

hydrochloric,   154,   199,   201 

lactic,    130,    145,    227 

oxalic,    212 

picric,  39 

propionic,  130 

pyrogallic    in    cultivating    anae- 
robes,   92 

rosolic,    79 
Acid-proof  bacilli,  43,  46,   149,   154, 

287,   295,   301 

Acids,  formation  by  bacteria,   130 
Actinomyces,  299,  229,  230,  287,  295 
Actinomycosis,    300 
Acquired   immunity,    176 
Active   immunity,    181 
Acute    miliaiy    tuberculosis,    293 
Addiment,    188 

Aerobic    bacteria,    definition,    126 
Aerobioscope,    136 
Agar-agar,  76 

Age,  relation  to  infections,   166 
Agglutinating    substances    in    blood- 
serum,   191,   305 
Agglutinins,  191,  305 
Air,  bacteria  of,  135 

bacteria  conveyed  by,  163 
Albumen,    culture-media    containing, 
81 


Albumen,  fixative,  50 
Alcohol,   acid,   42,  45 

fixation  of  tissues  by,  48 

relation  to  infection,  166,  191 
A.exins,   188,   190 

Alimentary    canal,    bacteria    of,    154 
Alum  filter,   139 
Amboceptor,  188 
American  filtration  system,   139 
American   Public   Health   Ass'n,   di- 
rections for  preparing  media,  73 
Amoeba  coli,  328,  329 

of  dysentery,  328 
Anaerobic    bacteria,    cultivation,    91 

definition,  126 
Aniline    dyes,    39 

alcoholic    solutions,     39 
as  germicides,  201 
watery  solutions,   39 

oil,   41,   53 

-water   solutions,   41,   46 
Animals,  autopsies  on,   105 

care   of,    103,    104 

inoculation  of,  96,  103 
Anopheles,  165,  332 
Anthrax  bacillus    (see  also  Bacillus 
of  anthrax),  273 

protective    inoculation,    178,276 

symptomatic,    178 
Antiseptic,  definition,  194 
Antitoxic  unit,  286 
Antitoxins,    180,    184 

for    diphtheria,    285,    180,    186, 

272 

tetanus,  180,  186,  272 
Argentamin,   200 
Argonin,  200 
Argyrol,    200 

Arnold   steam    sterilizer,    64 
Arrow-poisons,   bacteria   in,    16,    133 
Arthritis,   242,  254,   260 
Arthrospore,    123 
Asiatic   cholera    (see   Cholera) 
Aspergillus    glaucus,    233 


337 


338 


INDEX. 


Autoclave,   68 

Auto-infection,   165 

Autopsies,  on  animals,   105 

bacteriological  examinations  at, 

105,   no 

disinfection  at,   105,   106,  209 
on  human  subjects,  no 

Avian    tuberculosis,    295 

•pABES-ERNST    bodies,    121 
\j     Bacilli,  branching  forms,  119 
Bacilli,  acid-proof,  43,  46,   149,   154, 

287,   295,   301 

Bacillus,   definition,    14,    118 
acidi  lactici,   Hueppe,  227 
acidophilus,    155 
aerogenes,  313,  155,  261 
capsulatus,  268,  133 
amylobacter,  223,   156 
anthracis,  273,  25,  133,  135,  163, 

164,   166,   183,  264 
bifidus,   155 
botulinus,    150 
buccalis    maximus,    230 
butyricus,    Hueppe,    224 

Prazmowski,   223 
capsule,  of  Pfeiffer,  261 
coli    communis,    309,    145,    146, 
150,    156,    175,    192,    261, 
3i3 
comparison      with      typhoid 

bacillus,    310 
in  water,   143,   144 
comma   of   cholera    (see    Spiril- 
lum of  cholera) 
cyanogenus,    226 
diphtheriae,    278,    115,    163,    184 
dysenteric,   313,    137 
edematis  maligni,  270,  133 
enteridis,    Gartner,    150,    313 
erythrosporus,  226 
fluorescens  liquefaciens,   222 

putidus,  222 
icteroides,    160 
Indicus,   223 
influenzas,   277 

Klebs-Loffler  (see  Bacillus  diph- 
theria) 
lactis    aerogenes,    313,    155,    261 

cyanogenus,    226 
leprae,  296,  287 
mallei,   297,    192 
megatherium,    224 


Bacillus,  mesentericus  vulgatus   (see 

also  Potato  Bacillus),  224 
mucosus  capsulatus,  260 
mycoides,    225 
Neapolitanus,  309 
of    anthrax,    273,    25,    133,    135, 

163,  164,  166,   183,  264 
of  blue  milk,  226 
of  bubonic   plague,   265 
of  chancroid,   260 
of  diphtheria,  278,  115,  163,  184 
of  Ducrey,   260 
of  dysentery,   313,   137 
of   Eberth,    301 
of  Emmerich,  309 
of  Escherich,  309 
of   Friedlander,   260 
of  glanders,   297,    192 
of  influenza,   277 
of   leprosy,    296,    287 
of    malignant    edema,    270,    133 
of    ozaena,    261 
of   rhinoscleroma,    261 
of   Shiga,   313,    137 
of  smegma,  44,  154 
of  soft  chancre,  260 
of  syphilis,  Lustgarten,  160 

Joseph  and  Piorkowsky,  160 
of  tetanus,  270,  96,  133 
of  typhoid  fever,  301,   137,   142, 

M4.   145 

of  Vincent,   229 
of    Xerosis,    283 
paracolon,    312 
paratyphoid,   312 
pest  is  buboniae,   265 
phlegmones  emphysematosae,  270 
phosphorescens   Indicus,  225 
pneumoniae,    Friedlander,    260 
prodigiosus,    223 
proteus,    264,    150,    192 
pyocyaneus,    262,    192 
pyogenes   fetidus,   309 
ramosus,   225 
subtilis  (see  also  Hay  bacillus), 

225 

tetani,  270,  96,   133 
tuberculosis,    287,    96,    161,    163 
in  milk,   148,   149 
staining    of,     43,     54,     149, 

287 
typhi     abdominalis     (B.     typho- 

sus),   301,    137,    142,   144,    145 


INDEX. 


339 


Bacillus,  vaginalis,  153 

violaceus,  223 

Bacteria,     acid-proof,     43,     46,     149, 
154,    287,    295,    301 

aerobic,    126 

anaerobic,  126 

cultivation  of,  91 

chlorophyll,  relation  to,  n,  125, 
132,   iS7_ 

chromogenic,    128 

cultivation   of,    84 

classification,    117 

definition,    13 

diseases  caused  by,  159 

distribution,     14,     133 

examination     with     the     micro- 
scope,  29 

ferments  formed  by,   128 

fluorescent,  128,  222,  262 

forms    of,    117,    118 

higher,   229 

in  disease,   157 

influence    of    electricity,    127 
of   oxygen,    126 
of  sunlight,    126 

microscopic  examination,   29 

motility,   124 

multiplication,    122 

non-pathogenic,    120,  221 

number  of  species,  221 

nutrition   of,    125 

of  air,   135,   163 

of  the  alimentary  canal,  154 

of  foods,  144,  164 

of  the  cranial  sinuses,  151 

of    the    gall-bladder,     151,    308, 
312 

of  the  intestines,   154,   155 

of  ice,  136,  144 

of  milk,   144,   164 

of  the  mouth,  152,  227,  228,  230, 
253,   ^61 

of  the  nasal  cavity,  152,  261 

of     the     normal     human     body, 

151 

of  the  skin,    151 
of  soil,  133,  164 
of  the  stomach,    154 
of   the   urethra,    153 
of  the  vagina,   153 
of  water,   136,   164 
pathogenic,   121,  235 
phosphorescent,   128 
29 


Bacteria,    products    of   growth,    128, 

146,    150,    172 
pyogenic,  237 
size,    14,    121 
staining,   39,   40 

in  tissues,  48,  51 
transmission    of    specimens    by 

mail,   no 

vegetative  forms,   122 
Bacterial    products,     128,     146,     150, 

172 

Bacteriolysis,  184,   187,   189 
Bacterium,    definition,    119 

coli    commune,    309,     145,    146, 

150,  156,  175,  192,  261,  313 
syncyanum,  226 
termo,  132,  264 
urese,   227 
Zopfii,  227 

Balsam,  Canada,  38,  40,  52 
Basic  aniline  dyes,   39 
Basophilic  granules,  51,  53 
Beef-tea,  71 
Beggiatoa,    229 
Beri-beri,    160 
Berkenfeld  filter,  69 
Bichloride  of  mercury  (see  Mercury, 

bichloride) 
Biedert's     method     for     examining 

sputum,    47 

Birds,   tuberculosis  of,   295 
Bismarck  brown,  39,  42 
Black  death,   268 

leg,  178 

Blastomycetic  dermatitis,  233 
Blood-agar,   81,   277 
Blood,  cultures   from,    109 

of    another     species,     immunity 

for,  1 88,  192 
-poisoning,  239 
specimens  of,  108 
-serum-agar,   80 

germicidal    power,    190 

Loffler,  80 

Marmorek,   81,   249 

preparation,    79 

sterilization,  67,  79 

-test  for  typhoid  fever,  304, 

191 

staining    of,    55 
Blue  milk,  bacillus   of,   226 
pus,  263 
vitriol,  208 


340 


INDEX. 


Bodily    conditions    disposing    to    in- 
fection, 165 

Boiling,    sterilization    by,     63,     139, 
21 1,  214 

Boils,   245 

Bodphilus,   165 

Boric  acid,   207 

Bouillon,    71 

sugar-free,  74 

Bovine  tuberculosis,  148,  290,  294 

Branching   forms   of  bacilli,    119 

Bread-paste,   81 

Bromine,  as  a  germicide,  205 

Bronchitis,  241,  278 

Brownian   movement,    35 

Bubonic  plague,  bacillus,  265 

Buchner's     method     for     cultivating 
anaerobes,  91 

Butter,  tubercle  bacilli  in,   148,   149, 

44 
Butyric  acid,   130,   224 

/CADAVER,    care    of,    in    contag- 
\_^     ious  diseases,  209 

Calcium    compounds    as    germicides, 
205,  206 

hypochlorite,   205 
Canada   balsam,   38,  40,  52 
Capaldi's    culture-medium,    304 
Capsule  bacillus  of  PfeitTer,  261 
Capsules  of  bacteria,   121,  57 

staining  of,   57 
Carbol-fuchsin,  45 
Carbolic  acid,  200,  129,  197,  220 
Carbon   dioxide,    130 
Carbuncles,  245 
Carmine,   54,  55 
Caries  of  the  teeth,  153 

ition,  292 

Catgut,   surgical   preparation,  215 
(  'edar-wood  oil,  31 
Celloidin    imbedding,   48 
Cells,  epithelioid,  291 

giant,    291 

pus,    236 

Cellulitis,   _'47,    270 
Cellulose,     decomposition     by     bac- 
teria,   129,    156 

Centrifuge    for    milk   separator,    147 
Cerebro-spinal   meningitis,    _>5; 
Chancroid,   bacillus   of,   260 
Charbon  (see  Anthrax) 
Cliarbon  sytnptomatiqiic,  178 


Cheese-poisoning,    146 
Chemotaxis,    124 
Chicken-pox,    160 
Chloride  of  lime,  205 
Chlorine,   as   a  germicide,   205 
Chloroform,    as    a    preservative,    80 
Chlorophyll,  relation  to  bacteria,  n, 

125.   132,   157 
Cholera,   diagnosis,   321 

infantum,   265,   315 

nostras,   325 

-red  reaction,   318 

spirillum   (see  also  Spirillum  of 

cholera),   315 
Chromicized    catgut,    217 
Chromogenic   bacteria,    128 
Cladothrix,  229 
Classes    in    bacteriology,    hints    for 

teaching,   112,   113,    114,    115 
Classification  of  bacteria,   117 
Cleaning   fluid,   36 
Climate,  influence  on  infections,  166 
Clostridium,   definition,    i_>4 

butyricum,  223 
Coal-oil,    207 

Coccus,  definition,  14,  118 
Collodion,  48 

capsules,    106 
Colon    bacillus     (see    also    Bacillus 

coli  communis),  309 
contrasted      with      typhoid 

bacillus,  310 
Colon  group,   312 
Colonies  of  bacteria,  97,  99,  101 
Comma  bacillus  of  cholera  (see  also 

Spirillum  of  cholera),  315 
Comma-shaped    bacteria,    118,    120 
Complement,    188 
Condenser,  Abbe,  31 
Conidia.   -233,   299 
Conjunctivitis,    gonorrheal,    260 
Consumption,   293 
Contagious  disease,  definition,   158 

disinfection   after,   210 
Contrast-stains,  39,  42,  43,  47,  54 
Copperas,  207 
I  opper   sulphate,   208 
Cornet    forceps,    36,    37 
Corrosive     sublimate,     see     Mercury 

bichloride 
Cotton  plugs  for  tubes,  etc.,  23,  82, 

Cotton,  absorbent,  82,  92,  219 


INDEX. 


341 


Cover-glasses,  36 
Cover-glass    forceps,    36,    37 

preparations,   36,   37,   39 
Cow-pox,  21,  177 
Cranial    sinuses,   bacteria   of,    151 
Cream,   ripening,   148 
Creolin,   201 
Cresol,   201 

Croup,  membranous,  284 
Cultivation  of  anaerobic  bacteria,  QI 

of  bacteria,  84 
Culture-media,    definition,    17 

neutralization,  71,  72,  73 
preparation,    71 
reaction  of,  71,   74,   126 
sterilization,  63,  71,  74,  79, 

83 
-tubes,  82 

inoculation  of,   84 
sterilization  of,  82 
Cultures  at  autopsies,  105,  no 
from    blood,    1 09 
destruction  of,  99,   115 
sealing  of,  91 
Cumol,    216 

Cutting  of  sections,  50 
Cupric  sulphate,  208 
Cystitis,  241,  264,  308,  312,  313 
Cytase,    188 

DELAFIED'S  hematoxylin,  54 
Deneke's   spirillum,   325 
Dengue,    160 
Dental  caries,  153 
Deodorizers,    194 
Dermatitis,   blastomycetic,   233 
Dextrose,   74 

-agar,   77 

-bouillon,   74 

media  for  anaerobes,   91 
Diagnosis  of  actinomycosis,  301 

of  bubonic  plague,   266 

of  cholera,   321 

of  diphtheria,  280,  247 

of  dysentery,  315,  329 

of  glanders,  298 

of  gonorrhea,   258 

of  influenza,  278 

of  malaria,   330 

of  Malta  fever,  256 

of     meningitis,      cerebro-spinal, 
257 

of  pneumonia,  254 


Diagnosis    of   tuberculosis,    287,    44, 

294,  295 

of  typhoid  fever,  304,  307,  308 
Dilution-cultures,  99 
Diphtheria,  284,  247 

antitoxin,    285,     180,     186,    272 
bacillus,   278,    115,    163,   184 
diagnosis,   280,   247 
toxin,   174,   186,  284,  285 
Diphtheritic  inflammation    (see  also 
Pseudamembranous  inflamma- 

tion),  247,   282,  284 
Diplococcus,  definition,  118 

intracellularis    meningitidis,  256 
of  gonorrhea,  258,   183 
of  pneumonia   (see  also   Micro- 
coccus  lanceolatus),  251 
Disease,  bacteria  in,   157 
Diseases   caused   by  bacteria,    159 

by  protozoa,  328 
probably     due     to     microorgan- 
isms, 160 
infectious,    recovery    from,    171, 

176,   189 

Disinfectant,  194 
Disinfection   at  autopsies,    105,    106, 

209 

of  cultures,  99,  115 
of    dejecta,    208 
of  hands,  212 

of  houses,  209,  201,  204,  206 
of  sputum,  45,   208 
of   stools,   208 
of  test-tubes,  99,  115 
of   urine,    208 
surgical,  211,  220 
Distribution  of  bacteria,  133 
Dorset's  egg-medium,   81 
Dressings,  surgical  preparation,   219 
Drinking  water,  sterilization  of,  139 
Drying,    influence   on    bacteria,    123, 

125 

Ducrey's  bacillus,  260 
Dunham's  peptone  solution,  79 
Dyes,   aniline,    39 

as  germicides,  201 
Dysentery,    263,    314 
amoebic,  328 
bacillus,  313 

EAR,  middle,  bacteria  of,  151,  241 
Eberth's  bacillus  (see  also  Ba- 
cillus of  typhoid  fever),  301 


342 


INDEX. 


Edema,  malignant,  bacillus,  270,  133 
Egg-albumen    as    a    culture-medium, 

81 

Egg-medium  of  Dorset,  81 
Eggs,  in  cultivating  anaerobes,  81,  95 
Ehrlich's    side-chain    theory,     184 
Electricity,     influence     on     bacteria, 

127 

Eisner's    culture-medium,    304 
Emmerich's  bacillus,   309 
Emphysematous  gangrene,  269 
Endocarditis,  241,  245,  247,  254,  260 
Endogenous  spores,   123 
Enzymes,   128,  174 
Eosin,   39,   42 
Epithelioid  cells,  291 
Erysipelas,  250 
Escherich's   bacillus,   309 
Esmarch's  method  for  anaerobes,  95 

roll-tubes,   99 

Essential  oils  as  germicides,  207 
Eye-piece,    29,   30 

FALLOPIAN    tube,    bacteria    of, 
151 

tarcy-buds,   297 

Fat  in  culture  media,  81 

Fats,  decomposition  by  bacteria,  129 

Feces,  bacillus  of  tetanus  in,  271 

bacteria  of,   155 

disinfection,    208 

typhoid  bacilli,  examination  for, 

304,    307 
Fermentation,  22,  131 

-tube,  130 

Ferments,   development  by  bacteria, 
128 

and  toxins,  1 74 
Ferrous   sulphate,    207 
Fibrin,   Weigert's   stain,   53 
Film-preparations,  37,  38,  39 
Filter,    alum,    139 

American,  139 

Berkenfeld,  69 

infusorial  earth,  69 

Kitasato,  69 

mechanical,    139 

Pasteur-Chamberland,  69,  139 

sand,    138 

unglazed  porcelain,  69,   139 
Filtration,    sterilization   by,    69,    139 

of  water,  138 
Finkler  and  Prior  spirillum,  324 


Fishing    from    colonies,    101 
Fission  of  bacteria,   13 
Fixation  of  cover-glass  preparations, 
37,    39 

of  slide-preparations,  38,   39 

of    tissues,    48 
Flagella,  124 

staining,    58 
Flies,  bacteria  carried  by,   164,  309, 

321 

Fluid  for  cleaning,  36 
Fluorescence   of   bacteria,    128,    222, 

262 

Focusing  the  microscope,   32,   35 
FomI  es,  definition,   159 
Foods,  bacteria  of,  144,  149 

poisoning  by,  146,  150 
Food  used  by  oacteria,  125 
Foot  and  mouth  disease,  161 
Forceps,   Cornet,  36,  37 

cover-glass,   36,   37 

for  slides,   Kirkbride,   38 

Stewart,  36,  38 

Formaldehyde   as   a   germicide,   201, 
197,    210 

catgut,   216 

disinfection  of  rooms,  210 

fixation  of  tissues  with,  48 
Formalin    (see  Formaldehyde) 
Formic  acid,   130 
Fowl-cholera,  protective  inoculation, 

178 

Fowls,  tuberculosis  of,  295 
Fractional  sterilization,  63 
Frankel's  method  for  anaerobes,  93 

pneumococcus    (see  also  Micro- 
coccus  lanceolatus),  251 
Freeman's  pail  for  pasteurizing,  67, 

M7 

Freezing,    influence   on   bacteria.  144 
Friedlander's  bacillus  of  pneumonia, 

260 
Fuchsin,   39,   40 

acid,   39 

Fiirbringer's    method    for    disinfec- 
ting   hands,    212 
Fusiform  bacillus  of  Vincent,  229 

/~*  ABBETT'S  method  for  staining 
VJJ"     tubercle  bacilli,  46,  154 
Gall-bladder,    bacteria   of,    151,    308. 

312 
Gangrene,  emphysematous,  269 


INDEX. 


343 


Gas-burner,  Koch's,  90 

formation  by  bacteria,  130 

-phlegmons,  269 

-regulator,   89 

Gastric  juice,  germicidal  power,  154 
Gauze,  sterilization  of,  219 
Gelatin,   74 

liquefaction,   128 

tetanus  bacilli  in,  272 
Gelose,   see   agar-agar,    76 
Gentian-violet,  39,  40 
Geppert's  test  for  germicides,  196 
Germicidal    power    of    blood-serum, 

190 

Germicide,  definition,  194 
Germicides,  tests   for,   195 
Germ,  use  of  the  word,  13 
German  measles,   160 
Giant-cell,  291 
Glanders,  bacillus,  297,  192 

Straus'   method   for   diagnosing, 

298 

Glass  plates,    100 

Glassware,    sterilization    of,    61,    62 
Gloves,   rubber,   214 
Glucose  (see  also  Dextrose),  74 
Glycerin-agar,  77 

-albumen,    50 
Glycerin-bouillon,    74 
Gonococcus  of  Neisser,  258,   183 
Gonorrhea,  258,  240,  260 

diagnosis,    258 
Gram's    method,    41,    52 

bacteria  stained  by,  42 
not  stained  by,  43 
Gram-Giinther   method,    42 
Gram-Weigert   method,    53 
Gray  tubercle,  292 
Green  pus,  263 
Ground- water,     137 
Groups   of   bacteria,    118 
Guarnieri's  medium,   81 
Gun-cotton,  48 

Giinther's     modification     of     Gram's 
method,  42 

HAFFKINE'S     inoculations      for 
plague,    267,    179 

Hair-follicles,  infection  around,  239 
Hands,   disinfection,   212 
Hanging-block,   36 
Hanging-drop,    34 
Haptophore,    185 


Hardening  of  tissues,  48 

Hay    bacillus,    225,    113,    123,    146, 

i95 
Heat,  effect  on  growth  of  bacteria, 

125 

Heat,  sterilization  by,  61,   211 
Hematoxylin,  54 
Hematozoon    of    malaria,    330 
Higher  bacteria,    229 
Hill's  test  for  germicides,   195 
Hiss,    medium    of,    304 
Hiss,  stain  for  capsules,  57 
Historical  sketch  of  bacteriology,  18 
Hog   cholera,    180 
Holmes,    O.    W.,   22 
Honing   of  knives,    51 
Horse-hair,  surgical    preparation,  218 
Hot-air   sterilizer,  62 
Houses,   disinfection,   209,   201,  204, 

206 

Hueppe's  method  for   anaerobes,  95 
Hydrochloric  acid,   154,   199,  201 
Hydrogen,   cultivation   of  anaerobes 
under,  93 

peroxide,   206 

sulphide,    130 
Hydrophobia,    179,    160 

preventive  inoculation,    179 
Hypha,  233 

Hypochlorite  of  calcium,  205 
Hypodermic  inoculation  of  animals, 
104,    105 

ICE,   bacteria   of,    136,    144 
Ice-cream  poisoning,   146 
Illumination  for  the  microscope,  32 
Imbedding,  48 
Immune-body,    188 
Immunity,   176,  26 

acquired,    176 

active,    181 

antitoxic,    184 

bacteriolytic,    184 

natural,    176 

passive,   181 

racial,   177 

side-chain    theory,    184 

theories  of,    182 

unit,  286 

Impression-preparation,  37 
Incubator,  87,  88 
Indol,  129 

test  for,  129 


344 


INDEX. 


Infected  wounds,  220 
Infection,    bodily    conditions    favor- 
ing,  165 

local  conditions  favoring,  167 
of  investigators  with  pathogenic 

bacteria,   115 
of  wounds,   167,   169 
mixed,   169 

secondary,  169,  170,  240 
terminal,   169,   191 
Infectious   disease,   definition,    158 
Inflammation,    235,    240 

diphtheritic,      see      also      Pseu- 
domembranous     inflammation, 
247,    282,   284 
Influenza    bacillus,    277 
Infusorial  earth  in  filters,  69 
Inoculation  of  animals,  103 

in  isolating  bacteria,  96 
of  tube-cultures,  84 
Inoculations,  preventive,  177 
for  anthrax,  178,  276 
for   blackleg  of  cattle,    178 
for  bubonic  plague,  267,  179 
for  cholera,  320 
for  erysipelas  of  swine,  178 
fowl-cholera,   178 
for  hydrophobia,  179 
for   small-pox,   20,    177 
for  tuberculosis,  295 
for  typhoid   fever,   309 
Insects,    infections    spread    by,    164, 

165,   309,   321,   327,  332,   335 
Insects,   destruction  of,   204,  207 
Instruments,     surgical     preparation, 

21 i,  214 

Intermediary  body,  188 
Intermittent  sterilization,  63 
Intestine,  bacteria  of,  154,  155 
Intravenous  inoculation,   104 
Invisible  growth  on  potato,  303 

microbes,    121,    161 
Involution  forms  of  bacteria,  121 
Iodide   of  mercury,    199 
Iodine  solution,  42 
lodoform,  207 
Iris  diaphragm,   29 
Itch,   25 


J 


ENNER,  21 

Journals  of  bacteriology,    18 


KANGAROO      tendon,      surgical 
preparation   of,   217 
Kerosene,  207 

Kirkbride   forceps    for  slides,    38 
Kitasato    filter,    69 
Khitschpreparat,  37 
Klebs-Loffler    bacillus    (see    also    B. 

diphtheria),  278 
Knives,  sharpening  of,  51 
Koch,  26 
Koch's  gas-burner,  90 

method  for  anaerobes,  94 

plate-cultures,    26,    96, 

100 

rules,   158 

steam  sterilizer,  66,  67 
tests  for  germicides,  195 

T    ACTIC  ACID,   130,   145 
J_/    Lactose,  74 
Leeuwenhoek,  20 
Leprosy  bacillus,  296,  287 
Leptothrix,    229,   230 

buccalis,   230,    152 

innominate,  230 

maxima  buccalis,  230 
Leucin,   129 
Leucocytosis,    183 
Leucomaines,    1 73 
Ligatures,  surgical  preparation,  215, 

217,   218,   198 

Light,  influence  on  bacteria,  126 
Lime  as  a  germicide,  206 
Liquefaction  of  gelatin,  128 
Lister,  24 

Lithium-carmine,  55 
Litmus-agar,  77 

-milk,   79 

Lockjaw  (see  Tetanus) 
Loffler's  bacillus  of  diphtheria,   278, 
115,  163,   184 

blood-serum,  80 

methylene-blue,  41 

stain   for  flagella,  58 
Lump-jaw,  301 
Lungs,  bacteria  of  the,   151 
Lustgarten's     bacillus     of     syphilis, 

160 

Lymphoid    tissues,    relation    of    bac- 
teria to,    151,    162 
Lysins,   189 
Lysol,  201 


INDEX. 


345 


MADURA  disease,  Madura  foot, 
301 

Magnifying  power  of  objectives,  30 
Mails,  transmission  of  specimens  of 

bacteria  in,    no 

Malachite-green  as  a  germicide,  201 
Malaria,   165,  204,  207 

parasite    of,    330 
Malarial    parasite,    staining    of,    55, 

109 
Malignant  edema,  bacillus,  270,   133 

pustule,   276 
Mallein,  298 

Malta-fever,  micrococcus  of,  255 
Marmorek's  anti-streptococcus 

serum,    249 

serum-medium,    81,   249 
Massachusetts  steam  sterilizer,  66 
Mastzellen,  51 

Mayer's  glycerin-albumen,  50 
Measles,   160,  248,  284 
Meat,  tubercle  bacilli  in,  148 
Mechanical  filter,   139 
Medium,    culture-    (see    Culture-me- 
dium) 
Membranous  croup,  284 

rhinitis,  284 
Meningitis,  241,  254,  257,  261 

cerebro-spinal,  257 
Mercuric  chloride   (see  Mercury  bi- 
chloride) 
iodide,    199 
Mercurol,  200 
Mercury  bichloride,   199,   196,   197 

stock  solution,  199 
Metachromatic  granules  of  bacteria, 

121 

Metastatic  abscesses,  242 
Metchnikoff,  theory  of  phagocytosis, 

182,    188 
vibrio  of,  323 

Methyl    alcohol    lamp    in    formalde- 
hyde disinfection,  203 
Methylene-blue,  39,  40,  41 

as  a  germicide,  201 
Loffler's,  41 

Methyl-violet  as  a  germicide,  201 
Miasmatic  disease,  definition,  159 
Microbe,  use  of  the  word,  13 
Micrococcus,  definition,   14,   118 
agilis,   221 
amylovorus,   158 


Micrococcus,  gonorrheas,  258 

lanceolatus,  251 

melitensis,  255 

of  sputum  septicemia,  251 

Pasteuri,  251 

pneumonia  crouposae,  251 

pyogenes  tenuis,  255 

tetragenus,  250 

ureae,    221 

Micromillimeter,  32 
Micron,  u,  32 
Microscope,  29 

Microscopical    examination    of    bac- 
teria,  29 
Microtome,  50 
Miliary  tubercle,  292 

tuberculosis,  293 
Milk  as  a  culture-medium,  78 

bacteria  of,  144,   164 

number  of  bacteria  in,  147,  148 

pasteurization,  67,  146 

pathogenic  bacteria  in,   145 

-poisoning,   146 

samples  of,  108 

staining  bacteria  in,  149 

sterilization    in    infant    feeding, 
146 

tubercle  bacilli  in,  148,  149 

of  lime,  206 
Miller's  spirillum,  325 
Milzbrand  (see  Anthrax) 
Mixed  infection,  169 
Moisture,   effect   on   growth   of  bac- 
teria,  125 
Mosquitoes  as  carriers  of  infectious 

disease,   165,  332,  335 
Mosquitoes,  destruction  of,  204,  207 
Motility  of  bacteria,  35,   124 
Moulds,  231,   114,  135,  233 

cultivation,   81 
Mouth,  bacteria,   152,  227,  228,  230, 

253,   261 

Movement,  Brownian,  35 
Mucor  mucedo,  233 
Mucous  membranes,  bacteria  of,  151 

152 

Multiplication  of  bacteria,   122 
Mumps,    1 60 

Mustard   as   a   deodorizer,   207 
Mycelium,  233 
Mycetoma,  301 


346 


INDEX. 


NASAL   cavity,    bacteria   of,    152, 
361 

Natural  immunity,  176 
Necrosis  bacillus,  231 
Neisser's  gonococcus,  258,  183 

stain  for  diphtheria  bacilli,  278, 

279 
Neutral    red    in    culture-media,    77, 

303,  310,  313,  3i4 
Neutralization  of  culture-media,   71, 

72,    73 

Nitrate  of  silver,  200 
Nitrifying  bacteria,  130,  133 
Nitrogen  fixation  by  bacteria,  134 

liberation  by  bacteria,  130 
Nitroso-indol   reaction,   130 
Noma,  230 
Non-pathogenic   bacteria,    definition, 

120 

bacteria,  221 
Normal  solutions,  73 
Nose-piece,  29 

Novy's  method  for  anaerobes,  94 
Nucleins,  191 

Number  of  bacteria  in  feces,  155 
milk,   147,   148 
soil,    133 
water,  140 

species  of  bacteria,   221 
Nutrient   agar-agar,    76 
bouillon,  71 
gelatin,  74 
Nut.ition  of  bacteria,   125 

OBERMEIER'S  spirillum,  326 
Objectives,   29 
Ocular,  29 
Odors  developed  by  bacteria,  130 

from  water,   137 
Oese,  33 

Oidium  lactis,  233 
Oil,  aniline,  41,  53 

cedar-wood,    31 

culture-media      containing,      81, 
265 

-immersion  objective,  30,  31 
Oil,  kerosene,  207 
Oils,  essential,  as  germicides,  207 
Osteomyelitis,  240,  245,  308 
Otomycosis,   234 

Ovum,  bacteria  conveyed  in,  161 
Oxalic  acid,  _M  j 
Oxygen,  relation  of  bacteria  to,   126 


Oysters,  typhoid  fever  conveyed  by, 

150 

Ozaena,  bacillus,  261 
Ozone  in  purifying  water,   139 

PARACOLON    bacillus,   312 
Paraffin  imbedding,  49 
Faraform  or  paraformaldehyde,   202 
Parasite,  definition,   120 
Paratyphoid  bacillus,  312 
Park,  Roswell,  method  for  disinfect- 
ing hands,   212 
Park,  W.  H.,  method  for  cultivating 

anaerobes,  94 
Parietti's  method  for  examination  of 

water,   143 

Passive  immunity,  181 
Pasteur,  23,  26 

Pasteur-Chamberland   filter,   69,    1 39 
Pasteurization,    67,    146 
Pathogenic   bacteria,    definition,    IJT 
descriptions  of  species,  235 
Pear-blight,  16,  158 
Penicillium  glaucum,  233 
Peptone,  71,   129 

solution  concentrated,  323 
Dunham,    79 
Peptonizing     ferments     formed     by 

bacteria,    128 

Pericarditis,  241.  245.  247,  254 
Peritonitis,    241.    245,   247,    263,   265, 

312 

I'crlsucht,  290 

Permanganate  of  potassium,  207,  212 
Peroxide  of  hydrogen,  206 
Petri  dishes,  98 
Petroleum     for     destroying     insects, 

207 
Pfeiffer's  capsule  bacillus,  261 

reaction  for  cholera  spirillum 
(Pfeiffer's  phenomenon),  189, 
319 

Phagocytosis,  182,  188,  235.  334 
t'henol   (see  also  Carbolic  acid),  129 
Phenolthalein,  72 
Phosphorescence    of    bacteria,     128, 

225 

Picric    acid,    39 

Piorkowski's   culture-medium,   304 
Piroplasma,   165 
Placenta,          bacteria         transmitted 

through,    161 
Plague,  bubonic,  bacillus  of,  265 


INDEX. 


347 


Plants,  diseases  of,  16,  158 
Plasmodium  of  malaria,  330 
staining  of,  55,   109 
Plasmolysis,    121 
Plate-cultures,  96 
Platinum  wire,  33 

rules  for  use,  33,  85 
Pleuritis,  241,  245,  247,  254 
Pleuro-pneumonia  of  cattle,  161 
Plugs,  cotton,  for  tubes,  etc.,  82,  92 
Pneumococcus  of  Frankel   (see  also 

Micrococcus  lanceolatus),  251 
Pneumonia,  broncho-,  241,  247,  254, 

261,  263,  285,  301,  308 
croupous,  241,   253,   261 
diagnosis,  254,  255 
Pneumonomycosis,  233 
Poisoning  by  food,    146,    150 
Porcelain  filter,   69 
Post-mortems,    disinfection    at,    105, 

1 06,  209 

Post-office   rules    for   mailing   speci- 
mens of  bacteria,  no 
Potassium  permanganate,  207,  212 
Potato  as  a  culture-medium,  78 
invisible  growth  on,  303 
bacillus,   224,   95,    113,    123,   146 
Precipitins,  192 

Predisposition  to  infection,   166 
Products,    bacterial,    128,    146,    150, 

172 

Propionic  acid,   130 
Protargol,  200 
Protective  inoculation,  177 

for  anthrax,   178,  276 
for  blackleg  of  cattle,  178 
for  bubonic  plague,  267,  179 
for  Asiatic  cholera,  320 
for  erysipelas  of  swine,  178 
for   fowl-cholera,    178 
for   hvdrophobia   or   rabies, 

179 

for  small-pox,  20,  177 
for  tuberculosis,  295 
for  typhoid  fever,  309 
Proteus  mirabilis,   264 
vulgaris,  264 
Zenkeri,  264 

Protozoa,    pathogenic,    328,    27,    165 
Pseudo-gonococcus,  258 

-diphtheria  bacillus,  283 
-membranous          inflammations, 
247,  254,  282,  284,  315 

30 


Pseudo-pneumococcus,    255 

-tuberculosis,  296 
Ptomaine  poisoning,  150 
Ptomaines,    173 
Puerperal  fever,  21,  247,  284 
Pure  cultures,  25,  96,  101 
Pus,   blue,   263 

-cells,    236 

formation,   236 

green,    263 

samples    of,    108,    no 
Putrefaction,   23,    131 
Pyemia,   242 
Pyocyanin,   173,  262 
Pyogenic  bacteria,  237 
Pyoktanin,  201 
Pyosalpinx,    260 

Pyrogallic  acid  for  cultivating  anae- 
robes,  92 
Pyroxylin,  48 

QUARANTINE,  19 

<^ 

RABIES,  179,  160 
Racial  immunity,   177 

predisposition     to     infec- 
tion, 167 
Rats,  acid-proof  bacilli  of,  295 

relation  to  bubonic  plague,  267 
Ranschbrand,    178 
Ray-fungus  of  actinomycosis,  299 
Reactions   of  culture-media,    71,   74, 

126 

Receptor,    185 
Recovery    from    infectious    disease, 

171,   176,   189 

Reichert's  gas-regulator,  89 
Relapsing  fever,  spirillum,  326 
Rheumatic  fever,   160,  242 
Rheumatism,   160,  242 
Rhinoscleroma,  bacillus,  261 
Ricin,   174,  181 
Ripening  of  cream,   148 
Roll-tubes  of  Esmarch,  99 
Rooms,    disinfection,   209,   201,    204, 

206 

Root-tubercle  organisms,  134 
Rosolic  acid,  79 
Rougct,  178 
Rubber    caps    for    culture-tubes,    86, 

9i 
gloves,  214 


348 


INDEX. 


Rubber    stoppers    for    culture-tubes, 

86,   91,   92 
Rules  for  students,  115,  98,  99,  105, 

1 06 

of  Koch,  158 
of  post-office,  no 

SABOURAUD'S     culture-medium, 
82 

Saccharomyces  cerevisiae,  231 
Saccharose,  74 
Salt-agar,  266 
Sanarelli's  bacillus  of  yellow  fever, 

160 

Sand  filter,  138 
Sapremia,    171 

Saprophyte,  definition,   120,   157 
Sarcina,   118,  222 
pulmonum,   222 
ventriculi,  222,  155 
Sarcoma,     toxins     of     streptococcus 

for,  249 

Scarlet  fever,  160,  248,  249,  284 
Schatz's     method     for     disinfecting 

hands,   212 

Schizomycetes,  definition,  13 
Schultz's    method    for    neutralizing 

culture-media,  72 
Scrofula,   292 
Sclr^'cinerothlauf,  178 
Sealing  culture-tubes,  91 
Secondary  infection,  169,  170,  240 
Section-cutting,  50 
Sections,  staining  bacteria  in,  51 
carmine,  55 
Gram's  method,  52 
hematoxylin,   54 
tubercle  bacilli,  54 
Weigert  method,  53 
Sedgwick's  test  for  germicides,   197 

-Tucker  aerobioscope,  136 
Self-purification  of  water,  138 
Semen,  transmission  bacteria  by, 

161 

Semmelweis,  21 
Separator  for  milk,   147 
Septicemia,   171 
Serum  (see  Blood-serum) 

-test     for    typhoid    fever,     304, 

191 
Shiga's    bacillus    of    dysentery,    313, 

137 
Side-chain  theory  of  immunity,  184 


Silk   threads   in   testing   germicides, 
195 

surgical  preparation,  218 
Silkworm  gut,   surgical  preparation, 

218 
Silver,  germicidal  power  of,  218 

nitrate,  200 

wire  in  surgery,  218 
Sinuses,  cranial,  bacteria  of,  151 
Size    of    bacteria,    14,    121 
Skatol,   129 
Skin,  bacteria  of,   151 

disinfection,    152,    212,    213,  214 
Sleeping  sickness,   336 
Slides,  forceps  for,  38 

glass,   38 
Small-pox,  334,   177,  248 

inoculation  of,  20 
Smear-culture,  86 

preparations,  37,  38,  39 
Smegma  bacilli,  44,   154 
Snake- venom,    174,   188 
Sodium  hydroxide,  71,  73.  92 
Soft-chancre,  bacillus  of,  260 
Soil,  bacteria  of,  133,  164 
Solutions,  normal,   73 
Species  of  bacteria,   117 
Spirilla  in  the  mouth,   152,  227,  325 

in   water,    114,    142,   228,    326 
Spirillum,  definition,   14,   118,   120 

dentium,  227 

of    Asiatic    cholera,     315,     137, 
142,   145,   155,   189 

of   Deneke,   325 

of  Finkler  and  Prior,  324 

of  Metchnikoff,  323 

of  Miller,   325 

of  Obermeier,  326 

of   Vincent,    229 

plicatile,  229 

relapsing   fever,   326 

rubrum,  227 

rugula,   228 

sputigenum,   228 

tyrogenum,    325 

undula,  228 

volutans,  228 
Spirochaeta,   definition,    120 

dentium,   227 

Obermeieri,   326 

plicatile,   229 
Splenic  fever  (see  Anthrax) 

puncture   in  typhoid   fever,   307 


INDEX. 


349 


Sponges,  surgical  preparation  of,  218 
Spontaneous  generation,   13,  23 
Spores,   13,  24,   122 

arthro-,   123 

endogenous,   123 
Spores  of  the  malarial  parasite,  330, 

332 
Spores  of  moulds,  233 

resistance  to  heat,  etc.,  123 

staining,  56 

Sporotricha  or  sporothrix,  233 
Sputum,  collection,  44,   108 

disinfection,  45,   208 

staining,   44,    108,    287 
Stab-culture,  86 
Staining,  39,  40 

bacteria  in  tissues,  48,  51 

blood,  55 

capsules,   57 

diphtheria  bacillus,  278 

flagella,  58 

gonococcus,  258 

Gram's  method,  41,  52 

malarial  parasite,  55,  109 

sections,  51 

spores,  56 

tubercle  bacillus,  43 

in  milk,  44,   149 

sputum,      44,      45, 

1 08 

tissue,  54 
Stalactite  growth  of  plague  bacillus, 

265,  266 
Staphylococcus,  definition,  118 

cereus  albus,  237 
flavus,  237 

epidermidis  albus,  246,  147,  152 

pyogenes  albus,  245 

aureus    (see    also    Suppura- 
tion), 243,   148 

pyogenet,  citreus,  237 
Steam  sterilization,  63,  211 
Stegomyia,   165,   335 
Sterilization,   63,   211 

after  autopsies,  105,  106,  209 

by  the  autoclave,  68 

by  boiling,  63,   139,  211,  214 

by   filtration,   69,    139 

by  steam,   63,   211 

by  the  naked  flame,   61 

fractional,  63 

hot-air,  61 


Sterilization,  intermittent,  63 

of  blood-serum,  67,  79 

of    culture-media,    63,    71,    74, 
79,  83 

of  cultures,  99,  115 

of  dressings,  219 

of  glassware,  61,  62 

of  gloves,  rubber,  214 

of   hands,   212 

of  instruments,  214 

of  ligatures,  215,  217,  218 

of  milk  in  infant  feeding,   146 

of  test-tubes,  82 

of  water,   139 

steam,  63,  211 
Sterilizer,  Arnold,  64 

hot-air,   62 

Koch,  66,  67 

Massachusetts,    66 

steam,  64 
Stern  berg's   bulbs,   no 

determination     thermal     death- 
point  of  bacteria,  125 

tests  for  germicides,   195 
Stewart's  forceps,  36,  38 
Stick-culture  (see  Stab-culture) 
Stitch-abscesses,  246 
Stoddart's  culture-medium,  304 
Stomach,  bacteria  of,  154 
Stools,  disinfection,  208 
Storage  of  water,  138 
Straus'     method     for     diagnosis     of 

glanders,  298 

Streptococcus,   definition,    118 
brevis,  246 

lanceolatus,   251 

longus,  246 

mucosus,  255 

of  erysipelas,   250 

pyogenes     (see     also     Suppura- 
tion), 246,  145,  170 

serum,  249 
Streptothrix,  229,  230,   301 

actinomyces,   299 

cuniculi,    231 
Stropping  knives,  51 
Substance  sensibilitrice,  188 
Sugar-free  bouillon,    74 
Sugars  in  culture-media,  74,  76,  77 
Sulphur,    use    in    disinfection,    204, 

210 

Sunlight,  influence  on  bacteria,  126 
Suppuration,  235 


350 


INDEX. 


Surgical  disinfection,  211,  220 

infection,   167,  238 
Surra,  335 

Swarming  islands,  264 
Swine  erysipelas,   178 
Symptomatic  anthrax,   178 
Syphilis,  160,  161 

Systematic  study  of  species  of  bac- 
teria,  112 

''T^EACHING  bacteriology,  sugges- 

JL       tions  for,  112,  113,  114,  115 

Tendons,   animal,   as   ligatures,    198, 

215 
Test-tubes,  82 

inoculation  of,  84 
manner  of  holding,  84 
plugs  for,  82,  92 
sealing  of,  91 
sterilization,  82 
Teeth,  bacteria  of,    153 

caries  of,  153 

Terminal  infections,   169,   191 
Tetanus  antitoxin,  180,  186,  2~  2 
bacillus,   270,  96,    133 
toxin,   174,   1 86,   187,   2-2 
Tetrad,   definition,   118 
Texas    fever,    165 

Thermal  death-point  of  bacteria,  de- 
termination, 125 
Thermophilic  bacteria,    i  J5 
Thermostat    (see    Gas-regulator) 
Thiothrix,    229,    230 
Thread-reaction  of  bacteria,  192 
Thrush,  233 
Thymol,   108 
Tinea  favosa,  233 
Tinea   trichophytina.    j.$3 
Tissues,   fixation   and   hardening,   48 

staining   bacteria   in,   48,    51 
Titration  of  culture-media,  72,  ;.} 
Toxalbumens,   173 
Toxemia,  170 
Toxin,  definition,   173 
Toxins,    \-j2,   -2,    uo,    157,   180,    181, 

185 
of    diphtheria,     174,     186,    284, 

285 

of  tetanus,  174,  186,  187,  272 
Vrxophore,    185 
Trichophyton,  cultivation,  82 

rypanosome,   335,   192 
Tsetse-fly  disease,  336,   165 


Tubercle,  gray,  miliary,  yellow,   292 
structure,   290,   291 
bacillus,    287,    96,    161,    163 
in  butter,  44,  148,  149 
in  meat,   148 
in  milk,   44,   148,    149 
staining,    43,    54,    149,    289 
in  milk,  44,   149 
in  sputum,  44,  45,   108 

287 
in    sections   of   tissue;, 

54 
Tuberculin,  294 

R.,  295 
Tuberculosis,    292 

acute   miliary,    293 
bovine,   148,  290,  294 
diagnosis,   287,   44,   294,   295 
frequency,    148,   292 
immunity,  295 
of  birds,  295 
organs  affected  by,  293 
pseudo-,  296 

spread  of,  in  the  body,  292,  293 
Typhoid    fever,    bacillus,    301,    137, 

142,   144,   145 

contrasted    with    colon    ba- 
cillus, 310 
fever  diagnosis,  304,  307,  308 

serum-test,    304,    191 
Typhus    fever,    160 
Tyrosin,    129 
Tyrotoxicon,    146 

TTLTRA-MICROSCOPIC      organ- 
\^j      isms,    i  _>  i ,    161 
Unit,   immunity,   286 

decomposition  by  bacteria,  129 
Urethra,  bacteria.  153 
Urethritis.  gonorrheal,  260 
Urinary    bladder,    bacteria    of    (see 

also  cystitis),    151 
Trine,    disinfection,    208 

samples,   108 

-serum-agar,    259 

typhoid  bacilli   in,  308 
Uterus,   bacteria   of,    151 

VACCIXATIOX.  21,  177 
and   tetanus,    j;_- 
Vaccinia,    parasites    in,    334 
Vagina,    bacteria    of,    153 
Vaginitis,  gonorrheal,  260 


INDEX. 


351 


Van  Ermengem's  method  for  stain- 
ing flagella,   59 

Vegetative  forms  of  bacteria,   122 
Venom  of  snakes,   174,  188 
Vibrio,  definition,  120 

aquatilis,  326 

Berolinensis,   326 

Metchnikovi,  323 

proteus,    324 

rugula,  228 

Schuylkiliensis,    326 
Vibrion  septique,  270 
Villemin,    22 
Vincent,  bacillus  of,  229 
Vinegar,  bacteria  in,  15 
Violet,  gentian-,  39,  40 

methyl,    201 
Virulence  of  bacteria,  126,  168 

WARMTH,  effect  on  growth  of 
bacteria,    125 
Water,    bacillus    coli    communis    in, 

143,   144 
bacteria    of,    136 

conveyed  by,   137,   164 
filtration,    138 
ground-,    137 

infections  carried  by,  137 
number  of  bacteria  in,  140 
pathogenic  bacteria  in,  137,  142 
purification  by  ozone,   139 
samples  of,   108,   139 
self-purification,    138 
spirilla  in,  114,  228,  326 
sterilization  of,   139 
storage  of,  138 
Watery  solutions  of  aniline  dyes,  39 


Weir's      method      for      disinfecting 

hands,    213 
Welch's  stain  for  capsules,  57 

hypothesis,   190 
Whooping-cough,   160 
Weigert's   stain   for  fibrin  and  bac- 
teria,  53 
Widal's  serum-test  for  typhoid  fever, 

304 
Wire  baskets,  83 

platinum,    33 

silver,  218 

Wolffhiigel  plate,  141 
Wounds,  infected,  220 

infection  of,  167,  238 

irrigation  of,  220 
Wool-sorters'  disease,  135,  276 
Wright's  stain  for  blood,  55 

method  for  anaerobes,  92 
Wurtz's  culture-medium,  303 
Wurzcl  bacillus,  225 

X-RAYS,  127 
Xerosis  bacillus,  283 
Xylol,   49,   52 

YEASTS,  231,  114,  135,  155 
Yellow    fever,    335,    160,    161, 

165,  204,  207 
Yellow  tubercle,  292 
Yersin's  serum  for  plague,  267 

ZIEHL'S   carbol-fuchsin,   45 
Zinc   chloride,   208 

sulphate,  208 
Zoogloea,   122 


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SUBJECT  INDEX. 


Gould's  Medical  Dictionaries,  -  Pages  12,  13 
Morris'  Anatomy,  New  Edition,  -  -  Page  4 
Compends  for  Students,  -  Page  27 


SUBJECT.  PAOB 

Alimentary  Canal  (see  Sur- 
gery)     24 

Anatomy    7 

Anesthetics    18,  19 

Autopsies  (see  Pathology)  20 

Bacteriology    8 

Bandaging  (see  Surgery) . .  24 

Blood,  Examination  of .  . .  8 

Brain 8 

Chemistry.     Physics    9 

Children,  Diseases  of 11 

Climatology 19 

Clinical   Charts 25 

Compends 27 

Consumption  (see  Lungs).  16 

Cyclopedia  of  Medicine. . .  13 

Dentistry 11 

Diabetes  (see  Urin.  Organs)  25 

Diagnosis 11 

Diagrams  (see  Anatomy)  .  8 

Dictionaries,   Cyclopedias.  12 

Diet  and  Food 13 

Disinfection   16 

Dissectors 7 

Ear    14 

Electricity    14 

Embryology 7 

Emergencies 24 

Eye   14 

Fevers 15 

Food   13 

Formularies 21 

Gynecology    15 

Hay  Fever 25 

Heart 15 

Histology 15 

Hydrotherapy 19 

Hygiene    16 

Hypnotism 8 

Insanity 8 

Intestines 23 

Latin,  Medical   (see  Phar- 
macy)    21 

Life  Insurance 19 

Lungs 16 

Massage    17 

Materia  Medica 17 

Mechanotherapy 17 

Medical  Jurisprudence. ...  18 


SUBJECT.  PAOK 

Mental  Therapeutics 8 

Microscopy 18 

Milk    8,10 

Miscellaneous 18 

Nervous  Diseases 19 

Nose    25 

Nursing 20 

Obstetrics 20 

Ophthalmology 14 

Organotherapy    18 

Osteology  (see  Anatomy).      7 

Pathology 20 

Pharmacy 21 

Physical  Diagnosis 11 

Physical  Training 17 

Physiology 22 

Pneumotherapy 19 

Poisons  (see  Toxicology)  .  .    18 

Practice  of  Medicine 22 

Prescription  Books  (Phar- 
macy)    21 

Refraction  (see  Eye) 14 

Rest    19 

Sanitary  Science 16 

Serum-Therapy 17 

Skin 23 

Spectacles  (see  Eye) 14 

Spine  (see  Nervous  Dis- 
eases)    19 

Stomach 23 

Students'   Compends 27 

Surgery  and  Surgical  Dis- 
eases    24 

Technological  Books 9 

Temperature  Charts 25 

Therapeutics    17 

Throat    25 

Toxicology 18 

Tumors  (see  Surgery) ....    24 

U.  S.  Pharmacopoeia 22 

Urinary  Organs 25 

Urine 25 

Venereal  Diseases 26 

Veterinary  Medicine.  .....   26 

Visiting  Lists,  Physicians'. 
(Send  Jar  Special  Circu- 
lar.) 

Water  Analysis 16 

Women,   Diseases  of 15 


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PERSHING.      Diagnosis  of  Nervous  and  Mental  Disease.     Illus- 
trated. $1-25 
REGIS.     Mental  Medicine.     Authorized  Translation  by  H.  M. 
BANNISTER.  M.D.  $2.00 

STEARNS.  Mental  Diseases.  With  a  Digest  of  Laws  Relating 
to  Care  of  Insane.  Illustrated.  Cloth,  $2.75;  Sheep,  $3.25 

TUKE.  Dictionary  of  Psychological  Medicine.  Giving  the 
Definition,  Etymology,  and  Symptoms  of  the  Terms  used  in 
Medical  Psychology,  with  the  Symptom?,  Pathology,  and 
Treatment  of  the  Recognized  Forms  of  Mental  Disorders. 
Two  volumes.  $10.00 

WOOD,  H.  C.     Brain  and  Overwork.  .40 

CHEMISTRY  AND   TECHNOLOGY. 

Special  Catalogue  of  Chemical  Books  tent  free  upon  application. 

ALLEN.  Commercial  Organic  Analysis.  A  Treatise  on  the 
Modes  of  Assaying  the  Various  Organic  Chemicals  and  Prod- 
ucts Employed  in  the  Arts,  Manufactures,  Medicine,  etc., 
with  Concise  Methods  for  the  Detection  of  Impurities,  Adul- 
terations, etc.  8vo. 

Vol.  I.    Alcohols,  Neutral  Alcoholic  Derivatives,  etc.,  Ethers, 
Vegetable  Acids,  Starch,  Sugars,  etc.     3d  Edition.     $4.50 
Vol.  II,  Part  I.     Fixed  Oils  and  Fats,  Glycerol,  Explosives, 
etc.     3d  Edition.  $3.50 

Vol.  II,  Part  II.  Hydrocarbons,  Mineral  Oils,  Lubricants, 
Benzenes,  Naphthalenes  and  Derivatives,  Creosote,  Phenols, 
etc.  3d  Edition.  $3.50 

Vol.  II,  Part  III.  Terpenes,  Essential  Oils,  Resins,  Camphors, 
etc.  3d  Edition.  Preparing. 

Vol.  Ill,  Part  I.     Tannins,  Dyes,  and  Coloring  Matters.     3d 
Edition,  Enlarged   and    Rewritten.      Illustrated.          $4.50 
Vol.  Ill,  Part  II.     The  Amines,  Hydrazines  and  Derivatives, 
Pyridine  Bases.     The  Antipyretics,  etc.     Vegetable  Alka- 
loids, Tea,  Coffee,  Cocoa,  etc.     8vo.     2d  Edition.     $4.50 
Vol.  Ill,  Part  III.    Vegetable  Alkaloids,  Non-Basic  Vegetable 
Bitter  Principles.     Animal  Bases,  Animal  Acids,  Cyanogen 
Compounds,  etc.     2d  Edition,  8vo.  $4.50 

Vol.  IV.  The  Proteids  and  Albuminou*  Principles.  2d 
Edition.  $4.50 

BAILEY  AND  CADY.     Qualitative  Chemical  Analysis.       $1.25 
BARTLEY.     Medical  and   Pharmaceutical  Chemistry.     A  Text- 
Book  for  Medical,  Dental,  and  Pharmaceutical  Students.   With 
Illustrations,  Glossary,  and  Complete  Index.    5th  Ed.    $3.00 

BARTLEY.  Clinical  Chemistry.  The  Examination  of  Feces, 
Saliva,  Gastric  Juice,  Milk,  and  Urine.  $1.00 

BLOXAM.  Chemistry,  Inorganic  and  Organic.  With  Experi- 
ments. 9th  Ed.,  Revised.  284  Engravings.  $6.00 

BUNGE.  Physiologic  and  Pathologic  Chemistry.  From  the 
Fourth  German  Enlarged  Edition.  $3.00 

CALDWELL.  Elements  of  Qualitative  and  Quantitative  Chem- 
ical Analysis.  3d  Edition,  Revised.  $1.00 


10  SUBJECT  CATALOGUE. 


CAMERON.     Soap  and  Candles.     54  Illustrations.  $2.00 

CLOWES  AND  COLEMAN.  Quantitative  Analysis.  6th  Edi- 
tion. 125  Illustrations.  $3.50 

COBLENTZ.     Volumetric  Analysis.     Illustrated.  $1.25 

CONGDON.  Laboratory  Instructions  in  Chemistry.  With 
Numerous  Tables  and  56  Illustrations.  $1.00 

GARDNER.  The  Brewer,  Distiller,  and  Wine  Manufacturer. 
Illustrated.  $1.50 

GRAY.  Physics.  Volume  I.  Dynamics  and  Properties  of 
Matter.  350  Illustrations.  $4.50 

GROVES  AND  THORP.     Chemical  Technology.     The  Applica- 
tion of  Chemistry  to  the  Arts  and  Manufactures. 
Vol.  I.  Fuel  and  its  Applications.     607  Illustrations  and  4 
Plates.  Cloth,  $5.00;  *  Mor.,  $6.50 

Vol.11.    Lighting.     Illustrated.        Cloth,  $4.00;  i  Mor.,  $5.50 
Vol.  III.  Gas  Lighting.  Cloth,  $3.50;  *  Mor.,  $4.50 

Vol.  IV.  Electric  Lighting.     Photometry. 

Cloth,  $3.50;  *  Mor.,  $4.50 

HEUSLER.     The  Chemistry  of  the  Terpenes.  $4.00 

HOLLAND.  The  Urine,  the  Gastric  Contents,  the  Common 
Poisons,  and  the  Milk.  Memoranda,  Chemical  and  Micro- 
scopical, for  Laboratory  Use.  6th  Ed.  Illustrated.  $1.00 

LEFFMANN.  Compend  of  Medical  Chemistry,  Inorganic  and 
Organic.  4th  Edition,  Revised.  .80;  Interleaved,  $1.00 

LEFFMANN.  Analysis  of  Milk  and  Milk  Products.  2d  Edition, 
Enlarged.  Illustrated.  $1.25 

LEFFMANN.  Water  Analysis.  For  Sanitary  and  Technic  Pur- 
poses. Illustrated.  4th  Edition.  $1.25 

LEFFMANN.  Structural  Formulae.  Including  180  Structural 
and  Stereo-Chemical  Formuhe.  12mo.  Interleaved.  $1.00 

LEFFMANN  AND  BEAM.  Select  Methods  in  Food  Analysis. 
Illustrated.  $2.50 

MUTER.  Practical  and  Analytical  Chemistry.  3d  American 
from  the  Ninth  English  Edition.  Revised  to  meet  the  re- 
quirements of  American  Students.  56  Illustrations.  $1.25 

OETTEL.     Exercises  in  Electro-Chemistry.    Illustrated.  .75 

OETTEL.     Electro-Chemical  Experiments.     Illustrated.          .75 

RICHTER.  Inorganic  Chemistry.  5th  American  from  10th 
German  Edition.  Authorized  translation  by  EDGAR  F.  SMITH, 
M.A  ,  PH.D.  89  Illustrations  and  a  Colored"  Plate.  $1.75 

RICHTER.  Organic  Chemistry.  3d  American  Edition,  trans- 
lated from  the  8th  German  by  EDQAR  F.  SMITH,  lllus.  2  vols. 
Vol.  1.  Aliphatic  Series.  625  pages.  $3.00 

Vol.  II.  Carbocyclic  Series.     671  pages.  $3.00 

ROCKWOOD.  Chemical  Analysis  for  Students  of  Medicine, 
Dentistry,  and  Pharmacy.  Illustrated.  $1.50 

SMITH.     Electro-Chemical  Analysis.     3d  Ed.  39  lllus.     $1.50 

SMITH  AND  KELLER.  Experiments.  Arranged  for  Students 
in  General  Chemistry.  4th  Edition.  Illustrated.  .60 

SUTTON.  Volumetric  Analysis.  A  Systematic  Handbook  for 
the  Quantitative  Estimation  of  Chemical  Substances  by 
Measure,  Applied  to  Liquids,  Solids,  and  Gases.  8th  Edition, 
Revised.  112  Illustrations.  $5.00 

SYMONDS.     Manual  of  Chemistry.     2d  Edition.  $2.00 

TRAUBE.     Physico-Chemical  Methods.    97  Illustration*.    $1.50 


MEDICAL  BOOKS.  11 


THRESH.  WaWr  and  Water  Supplies.  3d  Edition.  $2.00 

ULZER  AND  FRAENKEL.  Chemical  Technical  Analysis. 

Translated  by  Fleck.  Illustrated.  $1.25 

WOODY.  Essentials  of  Chemistry  and  Urinalysis.  4th  Edition. 

Illustrated.  $1.50 

***  Special  Catalogue  of  Books  on  Chemistry  free  upon  application. 

CHILDREN. 

HATFIELD.     Compend     of    Diseases    of     Children.     With    a 
Colored  Plate.    3d  Ed.    Just  Ready.       .80;  Interleaved,  $1.00 
IRELAND.     The   Mental  Affections   of   Children.     Idiocy,    Im- 
becility, Insanity,  etc.     2d  Edition.  $4.00 
POWER.     Surgical  Diseases  of  Children  and  their  Treatment 
by  Modern  Methods.     Illustrated.  $2.50 
STARR.     The  Digestive  Organs  in  Childhood.     The  Diseases  of 
the  Digestive  Organs  in  Infancy  and  Childhood.     3d  Edition, 
Rewritten  and  Enlarged.     Illustrated.  $3.00 
STARR.     Hygiene  of  the  Nursery.     Including  the  General  Regi- 
men and  Feeding  of  Infants  and  Children,  and  the  Domestic 
Management   of   the   Ordinary    Emergencies   of    Early   Life, 
Massage,  etc.     6th  Edition.     25  Illustrations.  $1.00 
SMITH.     Wasting  Diseases  of  Children.     6th  Edition.  $2.00 
TAYLOR  AND  WELLS.     The  Diseases  of  Children.     2d  Edition, 
Revised  and  Enlarged.     Illustrated.     8vo.                           $4.50 
"It  is  well  worthy  the  careful  study  of  both  student  and  prac- 
titioner, and  can  not  fail  to  prove  of  great  value  to  both.     We 
do  not  hesitate  to  recommend  it." — Boston  Medical  and  Surgical 
Journal. 

DIAGNOSIS. 

BROWN.  Medical  Diagnosis.  A  Manual  of  Clinical  Methods. 
4th  Edition.  112  Illustrations.  Cloth,  $2.25 

DA  COSTA.  Clinical  Hematology.  A  Practical  Guide  to  Exam- 
ination of  Blood,  with  Reference  to  Diagnosis.  6  Colored 
Plates,  48  other  Illustrations.  Cloth,  $5.00 ;  Sheep,  $6.00 

DOUGLAS.  Surgical  Diseases  of  Abdomen,  with  Reference  to 
Diagnosis.  20  Full-Page  Plates.  Just  Ready. 

Cloth,  $7.00  ;  Sheep,  $8.00 

EMERY.  Bacteriological  Diagnosis.  2  Colored  Plates  and  32 
other  Illustrations.  $1.50 

MEMMINGER.    Diagnosis  by  the  Urine.    2d  Ed.    24  Illus.    $1.00 

PERSHING.  Diagnosis  of  Nervous  and  Mental  Diseases. 
Illustrated.  $1.25 

STEELL.     Physical  Signs  of  Pulmonary  Disease.  $1.25 

TYSON.  Handbook  of  Physical  Diagnosis.  For  Students  and 
Physicians.  By  the  Professor  of  Clinical  Medicine  in  the  Uni- 
versity of  Pennsylvania.  Illus.  4th  Ed.,  Improved  and  En- 
larged. With  2  Colored  and  55  other  Illustrations.  $1.50 

DENTISTRY. 

Special  Catalogue  of  Dental  Books  tent  free  upon  application. 
BARRETT.     Dental    Surgery    for    General    Practitioners    and 
Students  of  Medicine  and  Dentistry.     Extraction  of  Teeth, 
ete.     3d  Edition.     Illustrated.  $1.00 


12  SUBJECT  CATALOGUE. 


BROOMELL.  Anatomy  and  Histology  of  the  Human  Mouth 
and  Teeth.  Second  Edition,  Revised  and  Enlarged.  337 
handsome  Illustrations.  Cloth,  $4.50;  Leather,  $5.50 

FILLEBROWN.      Operative  Dentistry.     Illustrated.  $2.25 

GORGAS.  Dental  Medicine.  A  Manual  of  Materia  Medica  and 
Therapeutics.  7th  Edition.  Cloth,  $4.00;  Sheep,$5.00 

GORGAS.  Questions  and  Answers  for  the  Dental  Student. 
Embracing  all  the  subjects  in  the  Curriculum  of  the  Dental 
Student.  Octavo.  $6.00 

HARRIS.  Principles  and  Practice  of  Dentistry.  Including 
Anatomy,  Physiology,  Pathology,  Therapeutics,  Dental  Sur- 
gery, and  Mechanism.  13th  Edition.  Revised  by  F.  J.  S. 
GOROAS,  M.D.,  D.D.S.  1250  Illus.  Cloth,  $6.00 ;  Leather,  $7.00 

HARRIS.  Dictionary  of  Dentistry.  Including  Definitions  of  Such 
Words  and  Phrases  of  the  Collateral  Sciences  as  Pertain  to  the 
Art  and  Practice  of  Dentistry.  6th  Edition,  Revised  and 
Enlarged  by  FERDINAND  J.  S.  GOROAS,  M.D.,  D.D.S. 

Cloth,  $5.00 ;  Leather,  $6.00 

RICHARDSON.  Mechanical  Dentistry.  7th  Edition.  Thor- 
oughly Revised  and  Enlarged  by  DR.  GEO.  W.  WARREN.  691 
Illustrations.  Cloth,  $5.00;  Leather,  $6.00 

SMITH.     Dental  Metallurgy.    2d  Edition.    Illustrated.  $2.00 

TAFT.     Index  of  Dental  Periodical  Literature.  $2.00 

TOMES.     Dental  Anatomy.     263  Illustrations.     5th  Ed.  $4.00 

TOMES.     Dental  Surgery.     4th  Edition.     289  Illus.  $4.00 

WARREN.  Compend  of  Dental  Pathology  and  Dental  Medicine. 
With  a  Chapter  on  Emergencies.  3d  Edition.  Illustrated. 

.80;  Interleaved,  $1.00 

WARREN.     Dental  Prosthesis  and  Metallurgy.     129  Illus.    $1.25 
WHITE.     The  Mouth  and  Teeth.     Illustrated.  .40 

DICTIONARIES.     CYCLOPEDIAS. 

GOULD.  The  Illustrated  Dictionary  of  Medicine,  Biology,  and 
Allied  Sciences.  Being  an  Exhaustive  Lexicon  of  Medicine  and 
those  Sciences  Collateral  to  it:  Biology  (Zoology  and  Botany), 
Chemistry,  Dentistry,  Pharmacology,  Microscopy,  etc.,  with 
many  useful  Tables  and  numerous  fine  Illustrations.  1633 
pages.  Fifth  Edition. 

Sheep  or  Half  Morocco,  $10.00;  with  Thumb  Index,  $11.00 
Half  Russia,  Thumb  Index,  $12.00 

GOULD.  The  Medical  Student's  Dictionary,  nth  Edition.  Il- 
lustrated. Including  those  Words  and  Phrases  generally  used 
in  Medicine,  with  their  Proper  Pronunciation  and  Definition, 
Based  on  Recent  Medical  Literature.  With  Table  of  Epq- 
nymic  Terms  and  Tests  and  Tables  of  the  Bacilli,  Micrococci, 
Mineral  Springs,  etc.,  of  the  Arteries,  Muscles,  Nerves,  Ganglia, 
Plexuses,  etc.  Eleventh  Edition.  Enlarged  and  illustrated 
with  a  large  number  of  Engravings.  840  pages. 

Half  Morocco,  $2.50:  with  Thumb  Index,  $3.00 
Flexible  Leather,  Burnished  Edges,  Thumb  Index,     3.50 


MEDICAL  BOOKS.  13 


GOULD.  The  Pocket  Pronouncing  Medical  Lexicon.  4th  Edi- 
tion. (30,000  Medical  Words  Pronounced  and  Denned.)  Con- 
taining all  the  Words,  their  Definition  and  Pronunciation, 
that  the  Medical,  Dental,  or  Pharmaceutical  Student  Gener- 
ally Cornea  in  Contact  with;  also  Elaborate  Tables  of  Epo- 
nymic  Terms,  Arteries,  Muscles,  Nerves,  Bacilli,  etc.,  etc.,  a 
Dose  List  in  both  English  and  Metric  Systems,  etc.,  Arranged 
in  a  Most  Convenient  Form  for  Reference  and  Memorizing. 
Fourth  Edition,  Revised  and  Enlarged.  838  pages. 
Full  Limp  Leather,  Gilt  Edges,  $1.00;  Thumb  Index,  $1.25 
145,000  Copies  of  Gould's  Dictionaries  have  been  sold. 

GOULD  AND  PYLE.  Cyclopedia  of  Practical  Medicine  and 
Surgery.  Seventy-two  Special  Contributors.  Illustrated.  One 
Volume.  A  Concise  Reference  Handbook  of  Medicine,  Sur- 
gery, Obstetrics,  Materia  Medica,  Therapeutics,  and  the  Vari- 
ous Specialties,  with  Particular  Reference  to  Diagnosis  and 
Treatment.  Compiled  under  the  Editorial  Supervision  of 
GEORGE  M.  GOULD,  M.D.,  Author  of  "An  Illustrated  Dictionary 
of  Medicine,"  etc.;  and  WALTER  L.  PTLB,  M.D.,  Assistant 
Surgeon  Wills  Eye  Hospital;  formerly  Editor  "International 
Medical  Magazine,"  etc.,  and  Seventy-two  Special  Contribu- 
tors. With  many  Illustrations.  Large  Square  8vo,  to  corre- 
spond with  Gould's  "Illustrated  Dictionary." 
Full  Sheep  or  Half  Mor.,  $10.00;  with  Thumb  Index,  $11.00 
Half  Russia,  Thumb  Index,  $12.00  net. 

GOULD  AND  PYLE.  Pocket  Cyclopedia  of  Medicine  and  Sur- 
gery. Based  upon  above  book  and  uniform  in  size  with 
"Gould's  Pocket  Dictionary." 

Full  Limp  Leather,  Gilt  Edges,  $1.00 
With  Thumb  Index,  $1.25 

HARRIS.  Dictionary  of  Dentistry.  Including  Definitions  of 
Such  Words  and  Phrases  of  the  Collateral  Sciences  as  Pertain 
to  the  Art  and  Practice  of  Dentistry.  6th  Edition,  Revised 
and  Enlarged  by  FERDINAND  J.  S.  GORGAS,  M.D.,  D.D.S. 

Cloth,  $5.00;  Leather,  $6.00 

LONGLEY.     Pocket  Medical  Dictionary.  Cloth,  .75 

TREVES  AND  LANG.     German-English  Medical  Dictionary. 

Half  Calf,  $3.25 

DIET  AND  FOOD. 

ALLEN.  Proteids  and  Albuminous  Principles.  An  analytical 
Study  of  Food  Products.  2d  Edition.  $4.50 

BURNETT.  Foods  and  Dietaries.  A  Manual  of  Clinical  Diet- 
etics, with  Diet  Lists  for  Various  Diseases,  etc.  2dEd.  $1.50 

DAVIS.  Dietptherapy.  Food  in  Health  and  Disease.  With 
Tables  of  Dietaries,  Relative  Value  of  Foods,  etc.  Set  Cohen, 
Physiologic  Therapeutics,  page  17. 

GREENISH.  Microscopical  Examination  of  Foods  and  Drugs. 
Illustrated.  Just  Ready.  $3.50 

HAIG.  Diet  and  Food.  Considered  in  Relation  to  Strength  and 
Power  of  Endurance.  4th  Edition.  $1.00 

LEFFMANN.     Select  Methods  in  Food  Analysis.     Illua.         $2.50 


14  SUBJECT  CATALOGUE. 


EAR  (see  also  Throat  and  Nose). 

BURNETT.     Hearing  and  How  to  Keep  It.     Illustrated.          .40 

HOVELL.     Diseases  of  the  Ear  and  Naso-Pharynx.     Including 

Anatomy  and  Physiology  of  the   Organ,   together  with  the 

Treatment  of  the  Affections  of  the  Nose  and  Pharynx  which 

Conduce  to  Aural  Disease.      128  Illustrations.     2d  Ed.     $5.50 

PRITCHARD.     Diseases  of  the   Ear.     4th   Edition,   Enlarged. 

Many  Illustrations  arid  Formulae.  In  Press. 

ELECTRICITY. 

BIGELOW.     Plain  Talks  on  Medical  Electricity  and  Batteries. 

With  a  Therapeutic  Index  and  a  Glossary.     43  Illustrations. 

2d  Edition.  $1.00 

HEDLEY.     Therapeutic  Electricity  and  Practical  Muscle  Testing. 

99  Illustrations.  $2.50 

JACOB  Y.     Electrotherapy.    2  volumes.    Illustrated.    See  Cohen, 

Physiologic  Therapeutics,  page  17. 
JONES.     Medical  Electricity.     3d  Edition.     117  Illus.         $3.00 

EYE. 

A  Special  Circular  of  Books  on  the  Eye  sent  free  upon  application. 

DARIER.     Ocular  Therapeutics.     Just  Ready.  $3.00 

DONDERS.  The  Nature  and  Consequences  of  Anomalies  of 
Refraction.  With  Portrait  and  lllua.  Half  Morocco,  $1.25 

PICK.  Diseases  of  the  Eye  and  Ophthalmoscopy.  Translated 
by  A.  B.  HALE,  M.D.  157  llius.  Cloth,  $4.50;  Sheep,  $5.50 

GOULD  AND  PYLE.  Compend  of  Diseases  of  the  Eye  and  Re- 
fraction. Including  Treatment  and  Operations,  and  a  Section 
on  Local  Therapeutics.  With  Formulae,  Useful  Tables,  a 
Glossary,  and  111  Illus.,  several  of  which  are  in  colors.  2d 
Edition,  Revised.  Cloth,  .80;  Interleaved,  $1.00 

GREEFF.  The  Microscopic  Examination  of  the  Eye.  Illus- 
trated. $1.25 

HARLAN.     Eyesight,  and  How  to  Care  for  It.     Illus.  .40 

HARTRIDGE.  On  the  Ophthalmoscope.  4th  Edition.  With 
4  Colored  Plates  and  68  Wood-cuts.  $1.50 

HARTRIDGE.  Refraction.  104  Illustrations  and  Test  Types. 
12th  Edition,  Enlarged.  $1.50 

HANSELL  AND  SWEET.  Treatise  on  Diseases  of  the  Eye. 
With  many  Illus.  drawn  by  special  artists,  etc.  In  Press. 

HANSELL  AND  REBER.  Muscular  Anomalies  of  the  Eye. 
Illustrated.  $1.50 

HANSELL  AND  BELL.  Clinical  Ophthalmology.  Colored 
Plate  of  Normal  Fundus  and  120  Illustrations.  $1.50 

HENDERSON.     Notes  on  the  Eye.     3d  Ed.    Just  Ready.    $1.50 

JENNINGS.  Manual  of  Ophthaimoscopy.  95  Illustrations  and 
1  Colored  Plate.  $1.50 

MORTON.  Refraction  of  the  Eye.  Its  Diagnosis  and  the  Cor- 
rection of  its  Errors.  6th  Edition.  $1.00 

OHLEMANN.  Ocular  Therapeutics.  Authorized  Translation, 
and  Edited  by  DR.  CHARLEB  A.  OLIVER.  $1.75 

PARSONS.  Elementary  Ophthalmic  Optics.  With  Diagram- 
matic Illustrations.  $2.00 


MEDICAL  BOOKS.  15 


PHILLIPS.     Spectacles     and     Eyeglasses.     Their     Prescription 

and  Adjustment.     3d  Edition.     52  Illustrations.  51.00 

SWANZY.     Diseases    of   the    Eye    and    Their    Treatment.     8th 

Edition,  Revised  and  Enlarged.     167   Illustrations,   1  Plain 

Plate,  and  a  Zephyr  Test  Card.     Just  Ready.  $2.50 

From  The  Medical  News. 

"Swanzy  has  succeeded  in  producing  the  most  intellectually 
conceived  and  thoroughly  executed  re*.sume*  of  the  science  within 
the  limits  he  has  assigned  himself.  As  a  'student's  handbook,' 
small  in  size  and  of  moderate  price,  it  can  hardly  be  equaled." 
THORINGTON.  Retinoscopy.  4th  Edition,  Carefully  Revised. 
Illustrated.  $1.00 

THORINGTON.     Refraction  and  How  to  Refract.     200  Illustra- 
tions, 13  of  which  are  colored.     2d  Edition.  $1.50 
WALKER.     Student's   Aid    in    Ophthalmology.     Colored    Plate 
and  40  other  Illustrations  and  a  Glossary.                              $1.50 
WORTH.     Squint :    Its  Causes,  Pathology,  Treatment.        $2.00 
WRIGHT.     Ophthalmology.     2d  Edition,  Revised  and  Enlarged. 
117  Illustrations  and  a  Glossary.  $3.00 

FEVERS. 

GOODALL  AND  WASHBOURN.  Fevers  and  Their  Treatment. 
Illustrated.  $3.00 

GYNECOLOGY. 

BISHOP.  Uterine  Fibromyomata.  Their  Pathology,  Diag- 
nosis, and  Treatment.  Illustrated.  Cloth,  $3.50 

BYFORD  (H.  T.).  Manual  of  Gynecology.  3d  Edition,  Revised 
and  Enlarged.  363  Illustrations.  $3.00;  Sheep,  $3.50 

DUHRSSEN.  A  Manual  of  Gynecological  Practice.  105  Illus- 
trations. $1.50 

FULLERTON.  Surgical  Nursing.  3d  Edition,  Revised  and 
Enlarged.  69  Illustrations.  $1.00 

LEWERS.     Diseases  of  Women.     146  Illus.     5th  Ed.  $2.50 

LEWERS.     Cancer  of  the  Uterus.     Just  Ready.  $3.00 

MONTGpMERY.  Practical  Gynecology.  A  Complete  Sys- 
tematic Text-Book.  2d  Edition,  Revised  and  Enlarged. 
With  570  Illus.  Just  Ready.  Cloth,  $5.00;  Leather,  $6.00 

ROBERTS.  Gynecological  Pathology.  With  127  Full-page 
Plates  containing  151  Figures.  $6.00 

WELLS.  Compend  of  Gynecology.  Illustrated.  3d  Edition, 
Revised  and  Enlarged.  Just  Ready.  .80;  Interleaved,  $1.00 

HEART. 

THORNE.  The  Schott  Methods  of  the  Treatment  of  Chronic 
Heart  Disease.  Fourth  Edition.  Illustrated.  $2.00 

HISTOLOGY. 

CUSHING.  Compend  of  Histology.  By  H.  H.  CTTBHINO,  M.D., 
Demonstrator  of  Histology,  Jefferson  Medical  College,  Phila- 
delphia. Illus.  Nearly  Ready.  .80;  Interleaved,  $1.00 

LAZARUS-BARLOW.  Pathological  Anatomy  and  Histology. 
Illustrated.  Just  Ready.  $6.50 


1«  SUBJECT  CATALOGUE. 

STIRLING.  Outlines  of  Practical  Histology.  368  Illustrations. 
2d  Edition,  Revised  and  Enlarged.  With  new  Illus.  $2.00 

STOHR.  Histology  and  Microscopical  Anatomy.  Edited  by 
A.  SCHAPER,  M.D.,  University  of  Breslau,  formerly  Demon- 
strator of  Histology,  Harvard  Medical  School.  Fifth  Amer- 
ican from  10th  German  Edition,  Revised  and  Enlarged.  353 
Illustrations.  $3.00 

HYGIENE. 

Special  Catalogue  of  Books  on  Hygiene  sent  free  upon  application. 

CANFIELD.  Hygiene  of  the  Sick-Room.  A  Book  for  Nurses 
and  Others.  Being  a  Brief  Consideration  of  Asepsis,  Anti- 
sepsis, Disinfection,  Bacteriology,  Immunity,  Heating,  Venti- 
lation, etc.  $1.25 

CONN.     Agricultural  Bacteriology.     Illustrated.  $2.50 

CONN.     Bacteriology  of  Milk  and  Milk  Products,     Illus.     $1.25 

COPLIN.  Practical  Hygiene.  A  Complete  American  Text- 
Book.  138  Illustrations.  New  Edition.  Preparing. 

HARTSHORNE.     Our  Homes.     Illustrated.  .40 

KENWOOD.  Public  Health  Laboratory  Work.  110  Illustra- 
tions and  3  Plates.  $2.00 

LEFFMANN.  Select  Methods  in  Food  Analysis.  53  Illustra- 
tions and  4  Plates.  $2.50 

LEFFMANN.  Examination  of  Water  for  Sanitary  and  Technical 
Purposes.  4th  Edition.  Illustrated.  $1.25 

LEFFMANN.  Analysis  of  Milk  and  Milk  Products.  Illustrated. 
Second  Edition.  $1.25 

LINCOLN.     School  and  Industrial  Hygiene.  .40 

McFARLAND.  Prophylaxis  and  Personal  Hygiene.  Care  of 
the  Sick.  See  Cohen,  Physiologic  Therapeutics,  page  17. 

NOTTER.  The  Theory  and  Practice  of  Hygiene.  15  Platea  and 
138  other  Illustrations.  Svo.  2d  Edition.  $7.00 

PARKES  AND  KENWOOD.  Hygiene  and  Public  Health.  2d 
Edition,  Enlarged.  Illustrated.  $3.00 

ROSENAU.     Disinfection  and  Disinfectants.     Illus.  $2.00 

STARR.  The  Hygiene  of  the  Nursery.  Including  the  General 
Regimen  and  Feeding  of  Infants  and  Children,  and  the  Domes- 
tic Management  of  the  Ordinary  Emergencies  of  Early  Life, 
Massage,  etc.  6th  Edition.  2.5  Illustrations.  $1.00 

STEVENSON  AND  MURPHY.  A  Treatise  on  Hygiene.  By 
Various  Authors.  In  three  octavo  volumes.  Illustrated. 

Vol.  I,  $6.00;  Vol.  II,  $6.00;  Vol.  Ill,  $5.00 

THRESH.     Water  and  Water  Supplies.     3d  Edition.  $2.00 

WILSON.  Handbook  of  Hygiene  and  Sanitary  Science.  With 
Illustrations.  8th  Edition.  $3.00 

WEYL.  Sanitary  Relations  of  the  Coal- Tar  Colors.  Authorised 
Translation  by  HKNRT  LEFFMANN,  M.D.,  PH.D.  $1.25 

LUNGS  AND  PLEURAE. 

KNOPF.    Pulmonary  Tuberculosis.     Its  Modern  Prophylaxis  and 

Treatment  in  Special  Institutions  and  at  Home.    Illus.    $3.00 

STEELL.     Physical  Signs  of  Pulmonary  Disease.     Illua.        $1.25 


MEDICAL  BOOKS.  17 

MASSAGE.     PHYSICAL  EXERCISE. 

OSTROM.  Massage  and  the  Original  Swedish  Movements. 
Their  Application  to  Various  Diseases  of  the  Body.  A  Manual 
for  Students,  Nurses,  and  Physicians.  Fifth  Edition,  En- 
larged. 115  Illustrations,  many  of  which  are  original.  $1.00 

MITCHELL  AND  GULICK.  Mechanotherapy.  Exercise,  Ortho- 
pedics, Massage,  Ocular  Corrections,  etc.  Illustrated.  See 
Cohen,  Physiologic  Therapeutics,  below. 

TREVES.     Physical  Education.     Its  Value,  Methods,  etc.        .75 

MATERIA  MEDICA  AND  THERAPEUTICS. 

BRACKEN.  Outlines  of  Materia  Medica  and  Pharmacology.  $2.75 
COBLENTZ.     The  Newer  Remedies.     Including  their  Synonyms, 
Sources,  Methods  of  Preparation,  Testa,  Solubilities,  Doses, 
etc.     3d  Edition,  Enlarged  and  Revised.  $1.00 

COHEN.  Physiologic  Therapeutics.  Methods  other  than  Drug- 
Giving  useful  in  the  Prevention  of  Disease  and  in  the  Treat- 
ment of  the  Sick.  Mechanotherapy,  Mental  Therapeutics, 
Suggestion,  Electrotherapy,  Climatology,  Hydrotherapy, 
Pneumatotherapy,  Prophylaxis,  Dietetics,  Organotherapy, 
Phototherapy,  Mineral  Waters,  Baths,  etc.  11  volumes,  8vo. 
Illustrated.  (Subscription.)  Cloth,  $27.50;  *  Mor.,  $38.50 

Special  Descriptive  Circular  will  be  sent  upon  application. 
GORGAS.     Dental  Medicine.     A  Manual  of  Materia  Medica  and 
Therapeutics.     7th  Edition,  Revised.  $4.00 

GROFF.     Materia  Medica  for  Nurses,  with  Questions  for  Self- 
Examination.     2d  Edition,  Revised  and  Improved.          $1.25 
HELLER.     Essentials  of  Materia  Medica,  Pharmacy,  and  Pre- 
scription Writing.  •      $1.50 
HEWLETT.     Serum-Therapy.  $1.75 
MAYS.     Theine  in  the  Treatment  of  Neuralgia,     i  bound.        .50 
POTTER.     Handbook  of  Materia  Medica,  Pharmacy,  and  Thera- 
peutics, including  the  Action  of  Medicines,  Special  Therapeu- 
tics, Pharmacology,  etc.,  including  over  600  Prescriptions  and 
Formulae.     9th  Edition,  Revised  and  Enlarged.     With  Thumb 
Index  in  each  copy.     Just  Ready.      Cloth,  $5.00;  Sheep,  $6.00 
"In  conclusion  we  may  add  that  Dr.  Potter's  Therapeutics 
covers  a  wider  field  than  many  books  which  bear  this  title     He 
discusses  a  good  many  drugs  which  are  rarely  employed,  and 
therefore  the  book  is  as  useful  to  one  who  wishes  to  look  for  un- 
usual information  as  it  is  to  him  who  wishes  a  handbook  for  ready 
reference  in  the  treatment  of  disease  as  he  meets  it  from  day  to 
day." — Therapeutic  Gazette. 

POTTER.  Compend  of  Materia  Medica,  Therapeutics,  and  Pre- 
scription Writing,  with  Special  Reference  to  the  Physiological 
Action  of  Drugs.  6th  Edition.  .80;  Interleaved,  $1.00 

MURRAY.     Rough  Notes  on  Remedies.     4th  Edition.  $1.25 

SAYRE.  Organic  Materia  Medica  and  Pharmacognosy.  An 
Introduction  to  the  Study  of  the  Vegetable  Kingdom  and  the 
Vegetable  and  Animal  Drugs.  Comprising  the  Botanical  and 
Physical  Characteristics,  Source,  Constituents,  and  Pharma- 
copeial  Preparations,  Insects  Injurious  to  Drugs,  and  Pharma- 
cal  Botany.  With  sections  on  Histology  and  Microtechnique, 
by  W.  C.  STBVBNS.  374  Illustrations,  many  of  which  are 
original.  2d  Edition.  Cloth,  $4.50 


18  SUBJECT  CATALOGUE. 

TAVERA.     Medicinal  Plants  of  the  Philippines.  $2.00 

WHITE  AND  WILCOX.  Materia  Medica,  Pharmacy,  Pharma- 
cology, and  Therapeutics.  5th  American  Edition,  Revised  by 
REYNOLD  W.  WILCOX,  M.A.,  M.D.,  LL.D.,  Professor  of  Clinical 
Medicine  and  Therapeutics  at  the  New  York  Post-Graduate 
Medical  School.  Cloth,  $3.00 ;  Leather,  $3.50 

"The  care  with  which  Dr.  Wilcpx  has  performed  his  work  is 
conspicuous  on  every  page,  and  it  is  evident  that  no  recent  drug 
possessing  any  merit  has  escaped  his  eye.  We  believe,  on  the 
whole,  this  is  the  best  book  on  Materia  Medica  and  Therapeutics 
to  place  in  the  hands  of  students,  and  the  practitioner  will  find  it 
a  most  satisfactory  work  for  daily  use." — The  Cleveland  Medical 
Gazette. 

MEDICAL  JURISPRUDENCE  AND 
TOXICOLOGY. 

REESE.  Medical  Jurisprudence  and  Toxicology.  A  Text-Book 
for  Medical  and  Legal  Practitioners  and  Students.  6th 
Edition.  Revised  by  HENRY  LEFFMANN.  M.D. 

Cloth,  $3.00;  Leather,  $3.50 
"To  the  student  of  medical  jurisprudence  and  toxicology  it  ia 

invaluable,  as  it  is  concise,  clear,  and  thorough  in  every  respect." 

— The  American  Journal  of  the  Medical  Sciences. 

MANN.     Forensic  Medicine  and  Toxicology.     Illus.  $6.50 

TANNER.  Memoranda  of  Poisons.  Their  Antidotes  and  Tests. 
8th  Edition,  by  DR.  HENRY  LBFFMANN.  Just  Ready.  .75 

MICROSCOPY. 

CARPENTER.  The  Microscope  and  Its  Revelations.  8th 
Edition.  Revised  and  Enlarged.  817  Illustrations  and  23 
Plates.  Cloth,  $8.00 ;  Half  Morocco,  $9.00 

GREENISH.  Microscopical  Examination  of  Foods  and  Drugs. 
Illustrated.  Just  Ready.  $3.50 

LEE.  The  Microtomist's  Vade  Mecum.  A  Handbook  of 
Methods  of  Microscopical  Anatomy.  887  Articles.  5th 
Edition,  Enlarged.  $4.00 

OERTEL.  Medical  Microscopy.  A  Guide  to  Diagnosis,  Ele- 
mentary Laboratory  Methods  and  Microscopic  Technic.  131 
Illustrations.  Just  Readjj.  $2.00 

REEVES.  Medical  Microscopy,  including  Chapters  on  Bacteri- 
ology, Neoplasms,  Urinary  Examination,  etc.  Numerous 
Illustrations,  some  of  which  are  printed  in  colors.  $2.50 

WETHERED.  Medical  Microscopy.  A  Guide  to  the  Use  of  the 
Microscope  in  Practical  Medicine.  100  Illustrations.  $2.00 

MISCELLANEOUS. 

BERRY.     Diseases  of  Thyroid  Gland.     Illustrated.  $4.00 

BUXTON.     Anesthetics.     Illustrated.     3d  Edition.  $1.50 

COHEN.     Organotherapy.    See  Cohen,  Physiologic  Therapeutics, 

page  17. 

FRENKEL.     Tabetic  Ataxia.     Illustrated.  $3.00 

GOULD.     Borderland    Studies.     Miscellaneous    Addresses    and 

Eosays.     12mo.  $2.00 


MEDICAL  BOOKS.  1» 

GOULD.  Biographic  Clinics.  The  Origin  of  the  Ill-Health  of 
DeQuincy,  Carlyle,  Darwin,  Huxley,  and  Browning.  Just 
Ready.  $1.00 

GREENE.     Medical    Examination    for    Life    Insurance.     Illus. 
With  colored  and  other  Engravings.     2d  Edition.       In  Press. 
HAIG.     Causation  of  Disease  by  Uric  Acid.     The  Pathology  of 
High  Arterial  Tension,  Headache,  Epilepsy,  Gout,  Rheuma- 
tism, Diabetes,  Bright's  Disease,  etc.     6th  Edition.         $3.50 
HENRY.     A  Practical  Treatise  on  Anemia.  Half  Cloth,  .50 

NEW  SYDENHAM  SOCIETY'S  PUBLICATIONS.  Circulars 
upon  application.  Per  Annum,  $8.00 

OSGOOD.     The  Winter  and  Its  Dangers.  .40 

PACKARD.     Sea  Air  and  Sea  Bathing.  .40 

RICHARDSON.     Long  Life  and  How  to  Reach  It.  .40 

SCHEUBE.  Diseases  of  Warm  Countries.  Illustrated.  Just 
Ready.  $8.00 

TISSIER.     Pneumotherapy,    Aerotherapy,  Inhalation  Methods. 

See  Cohen,    Physiologic  Therapeutics,  page  17. 
TURNBULL.     Artificial  Anesthesia.     4th  Ed.     Illus.  $2.50 

WARDEN.     The  Paris  Medical  School.  Paper,  .75 

WEBER  AND  HINSDALE.     Climatology  and  Health  Resorts. 
Including  Mineral  Springs.     2  vols.     Illustrated  with  Colored 
Maps.     See  Cohen,  Physiologic  Therapeutics,  page  17. 
WILSON.     The  Summer  and  Its  Diseases.  .40 

WINTERNITZ.  Hydrotherapy,  Thermotherapy,  Phototherapy, 
Mineral  Waters,  Baths,  etc.  Illustrated.  See  Cohen,  Physio- 
logic Therapeutics,  page  17. 

NERVOUS  DISEASES. 

DERCUM.  Rest,  Suggestion,  Mental  Therapeutics.  See  Cohen, 
Physiologic  Therapeutics,  page  17. 

GORDINIER.  The  Gross  and  Minute  Anatomy  of  the  Central 
Nervous  System.  With  271  original  colored  and  other  Illus- 
trations. Cloth,  $6.00;  Sheep,  $7.00 

GOWERS.     Syphilis  and  the  Nervous  System.  $1.00 

GOWERS.     Manual   of   Diseases   of   the    Nervous   System.     A 

Complete  Text-Book.     Revised,  Enlarged,  and  in  many  parts 

Rewritten.     With  many  new  Illustrations.     Two  volumes. 

Vol.  I.  Diseases  of  the  Nerves  and  Spinal  Cord.     3d  Edition, 

Enlarged.  Cloth,  $4.00;  Sheep,  $5.00 

Vol.  II.  Diseases  of  the  Brain  and  Cranial  Nerves ;  General  and 

Functional  Disease.     2d  Ed.          Cloth,  $4.00 ;  Sheep,  $5.00 

GOWERS.  Epilepsy  and  Other  Chronic  Convulsive  Diseases. 
2d  Edition.  $3.00 

HORSLEY.  The  Brain  and  Spinal  Cord,  the  Structure  and 
Functions  of.  Numerous  Illustrations.  $2.50 

ORMEROD.  Diseases  of  the  Nervous  System.  66  Wood  En- 
gravings. $1.00 

PERSHING.  Diagnosis  of  Nervous  and  Mental  Diseases.  Illus- 
trated. $1.25 

PRESTON.  Hysteria  and  Certain  Allied  Condition!.  Their 
Nature  and  Treatment.  Illustrated.  $2.00 

WOOD.     Brain  Work  and  Overwork.  .40 


20  SUBJECT  CATALOGUE. 


NURSING  (see  also  Massage). 

Special  Catalogue  of  Books  for  Nurses  sent  fret  upon  application. 

CANFIELD.  Hygiene  of  the  Sick-Room.  A  Book  for  Nurses 
and  Others.  Being  a  Brief  Consideration  of  Asepsis,  Anti- 
sepsis, Disinfection,  Bacteriology,  Immunity,  Heating  and 
Ventilation,  and  Kindred  Subjects  for  the  Use  of  Nurses  and 
Other  Intelligent  Women.  $1.25 

CUFF.     Lectures  to  Nurses  on  Medicine.     4th  Edition.        $1.25 

DAVIS.  Bandaging.  Its  Principles  and  Practice.  163  Original 
Illustrations.  $1.50 

DOMVILLE.  Manual  for  Nurses  and  Othert  Engaged  in  At- 
tending the  Sick.  9th  Edition.  With  Recipes  for  Sick-room 
Cookery,  etc.  In  Press. 

FULLERTON.     Obstetric  Nursing.     6th  Ed.   45   Illus.         $1.00 

FULLERTON.     Surgical  Nursing.     3d  Ed.     69  Illus.  $1.00 

GROFF.  Materia  Medica  for  Nurses.  With  Questions  for  Self- 
Examination.  2d  Edition,  Revised  and  Improved.  Just 
Ready.  $1.25 

HADLEY.  General,  Medical,  and  Surgical  Nursing.  A  very 
Complete  Manual,  Including  Sick-room  Cookery.  $1 .25 

HUMPHREY.  A  Manual  for  Nurses.  Including  General 
Anatomy  and  Physiology,  Management  of  the  Sick-room,  etc. 
24th  Edition.  79  Illustrations.  $1.00 

STARR.  The  Hygiene  of  the  Nursery.  Including  the  General 
Regimen  and  Feeding  of  Infants  and  Children,  and  the  Domes- 
tic Management  of  the  Ordinary  Emergencies  of  Early  Life, 
Massage,  etc.  6th  Edition.  25  Illustrations.  $1.00 

TEMPERATURE  AND  CLINICAL  CHARTS.     See  page  25. 

VOSWINKEL.  Surgical  Nursing.  Second  Edition,  Enlarged. 
112  Illustrations.  $1.00 

WILCOX.     Fever  Nursing.  Preparing. 

OBSTETRICS. 

CAZEAUX  AND  TARNIER.  Midwifery.  With  Appendix  by 
MuNDfc.  The  Theory  and  Practice  of  Obstetrics,  including  the 
Diseases  of  Pregnancy  and  Parturition,  Obstetrical  Operations, 
etc.  8th  Edition.  Illustrated  by  colored  and  other  full-page 
Plates,  and  numerous  Wood  Engravings. 

Cloth,  $4.50;  Full  Leather,  $5.50 

EDGAR.  Text-Book  of  Obstetrics,  By  J.  CLIFTON  EDGAK, 
M.D.,  Professor  of  Obstetrics  and  Clinical  Midwifery,  Medical 
Department  of  Cornell  University,  New  York  City.  etc.  1221 
Illustrations.  Just  Ready.  Cloth,  $6.00;  Sheep,  $7.00 

FULLERTON.     Obstetric  Nursing.     6th  Ed.      Illus.  $1.00 

LANDIS.  Compend  of  Obstetrics.  7th  Edition,  Revised  by 
WM.  H.  WILLS,  M.D.,  Demonstrator  of  Clinical  Obstetrics, 
Jefferson  Medical  College.  52  Illustrations. 

.80;  Interleaved,  $1.00 

WINCKEL.  Text-Book  of  Obstetrics,  Including  th«  Pathology 
and  Therapeutics  of  the  Puerperal  State.  Illustrated.  $5  00 

PATHOLOGY. 

BLACKBURN.  Autopsies.  A  Manual  of  Autopsies  Designed 
for  the  Use  of  Hospitals  for  the  Insane  and  other  Public  Insti- 
tutions. Ten  full-page  Plates  and  other  Illustration*.  $1.25 


MEDICAL  BOOKS.  21 


COPLIN.  Manual  of  Pathology.  Including  Bacteriology,  Tech- 
nic  of  Post-Mortems,  Methods  of  Pathologic  Research,  etc. 
330  Illustrations,  7  Colored  Plates.  3d  Edition.  $3.50 

DA  COSTA.  Clinical  Hematology.  A  Practical  Guide  to  the 
Examination  of  the  Blood.  Six  Colored  Plates  and  48  Illus- 
trations. Cloth,  $5.00 ;  Sheep,  $6.00 

LAZARUS-BARLOW.  Pathological  Anatomy.  With  7  Colored 
Plates  and  171  other  Illustrations.  $6.50 

MacLEOD.  The  Pathology  of  the  Skin.  Colored  and  other 
Illustrations.  Just  Ready.  $5.00 

MARTIN.     Manual  of  Pathology.     Illustrated.     Nearly  Ready. 

ROBERTS.     Gynecological  Pathology.     Illustrated.  $6.00 

THAYER.     Compend  of  General  Pathology.     Illustrated. 

.80;  Interleaved,  $1.00 

THAYER.     Compend  of  Special  Pathology.     Illustrated. 

.80;  Interleaved,  $1.00 

THAYER.  Manual  of  General  and  Special  Pathology.  Illus- 
trated. Nearly  Read}/. 

VIRCHOW.     Post-Mortem  Examinations.     3d  Edition.  .75 

WHITACRE.  Laboratory  Text-Book  of  Pathology.  With  121 
Illustrations.  $1.50 

PHARMACY. 

Special  Catalogue  of  Books  on  Pharmacy  sent  free  upon  application. 

COBLENTZ.  Manual  of  Pharmacy.  A  Complete  Text-Book  by 
the  Professor  iu  the  New  York  College  of  Pharmacy.  2d  Ed., 
Revised  and  Enlarged.  437  Illus.  Cloth,  $3.50;  Sheep,  $4.50 

COBLENTZ.     Volumetric  Analysis.     Illustrated.  $1.25 

BEASLEY.  Book  of  3100  Prescriptions.  Collected  from  the 
Practice  of  the  Most  Eminent  Physicians  and  Surgeons — Eng- 
lish, French,  and  American.  A  Compendious  History  of  the 
Materia  Medica,  Lists  of  the  Doses  of  all  the  Officinal  and  Es- 
tablished Preparations,  an  Index  of  Diseases  and  their  Reme- 
dies. 7th  Edition.  $2.00 

BEASLEY.  Druggists'  General  Receipt  Book.  Comprising  a 
Copious  Veterinary  Formulary,  Recipes  in  Patent  and  Pro- 
prietary Medicines,  Druggists'  Nostrums,  etc.;  Perfumery  and 
Cosmetics,  Beverages,  Dietetic  Articles  and  Condiments,  Trade 
Chemicals,  Scientific  Processes,  and  many  Useful  Tables. 
10th  Edition.  $2.00 

BEASLEY.  Pharmaceutical  Formulary.  A  Synopsis  of  the 
British,  French,  German,  and  United  States  Pharmacopoeias. 
Comprising  Standard  and  Approved  Formula  for  the  Prepara- 
tions and  Compounds  Employed  in  Medicine.  12th  Ed.  $2.00 

GREENISH.  Microscopical  Examination  of  Foods  and  Drugs. 
Illustrated.  Just  Heady.  $3.50 

ROBINSON.  Latin  Grammar  of  Pharmacy  and  Medicine.  4th 
Edition.  With  elaborate  Vocabularies.  Just  Ready.  $1.50 

SAYRE.  Organic  Materia  Medica  and  Pharmacognosy.  An 
Introduction  to  the  Study  of  the  Vegetable  Kingdom  and  the 
Vegetable  and  Animal  Drugs.  Comprising  the  Botanical  and 
Physical  Characteristics,  Source,  Constituents,  and  Pharma- 
copeial  Preparations,  Insects  Injurious  to  Drugs,  and  Phar- 
macal  Botany.  With  sections  on  Histology  and  Microtech- 
nique, by  W.  C.  STBVENS.  374  Illustration*.  Second  Edition. 

Cloth,  $4.50 


SUBJECT  CATALOGUE. 


SCOVILLE.  The  Art  of  Compounding.  Second  Edition,  Re- 
vised and  Enlarged.  Cloth,  $2.50 

STEWART.  Compend  of  Pharmacy.  Based  upon  "Reming- 
ton's Text-Book  of  Pharmacy."  5th  Edition,  Revised  in 
Accordance  with  the  U.  S.  Pharmacopoeia,  1890.  Complete 
Tables  of  Metric  and  English  Weights  and  Measures. 

.80;  Interleaved,  $1.00 

TAVERA.     Medicinal  Plants  of  the  Philippines.  $2.00 

UNITED  STATES  PHARMACOPOEIA.  7th  Decennial  Revision. 
Cloth,  $2.50  (postpaid,  $2.77) ;  Sheep,  $3.00  (postpaid,  $3.27) ; 
Interleaved,  $4.00  (postpaid,  $4.50) ;  Printed  on  one  side  of 
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PHYSIOLOGY. 

BIRCH.     Practical   Physiology.     An    Elementary    Class   Book. 

62  Illustrations.  $1.75 

BRUBAKER.  Text-Book  of  Physiology.  Illus.  Nearly  Ready. 
BRUBAKER.  Compend  of  Physiology,  llth  Edition,  Revised 

and  Enlarged.     Illustrated.  .80;  Interleaved,  $1.00 

JONES.     Outlines  of  Physiology.     96  Illustrations.  $1.50 

KIRKES.  Handbook  of  Physiology.  17th  Authorized  Edition. 
Revised,  Rearranged,  and  Enlarged.  By  PROT.  W.  D.  HALLI- 
BURTON, of  Kings  College,  London.  681  Illustrations,  some  of 
which  are  in  colors.  Cloth,  $3.00 ;  Leather,  $3.75 

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to  the  Requirements  of  Practical  Medicine.  5th  American, 
translated  and  edited  from  the  last  German  Edition  by  A.  P. 
BRUBAKER,  M.D.,  and  A.  A.  ESIINER,  M.D.  In  Press. 

STARLING.    Elements  of  Human  Physiology.     100  Illus.    $1.00 

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ical and  Experimental  Physiology,  with  Special  Reference  to 
Practical  Medicine.  3d  Edition.  289  Illustrations.  $2.00 

TYSON.     Cell  Doctrine.     Its  History  and  Present  State.     $1.50 

PRACTICE. 

BEALE.  On  Slight  Ailments  :  their  Nature  and  Treatment.  2d 
Edition,  Enlarged  and  Illustrated.  $1.25 

COHEN.  Physiologic  Therapeutics.  The  Treatment  of  Disease 
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FAGGE.  Practice  of  Medicine.  4th  Edition,  by  P.  H.  PYE- 
SMITH,  M.D.  2  volumes.  Vol.4,  $6.00;  Vol.  II,  $6.00 

FOWLER.     Dictionary     of     Practical     Medicine.     By     various 
of  Medicine. 
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MEDICAL  BOOKS.  28 


GOULD  AND  PYLE.  Cyclopedia  of  Practical  Medicine  and 
Surgery.  A  Concise  Reference  Handbook,  with  particular 
Reference  to  Diagnosis  and  Treatment.  Edited  by  DRS. 
GOULD  and  PTLE,  Assisted  by  72  Special  Contributors.  Illus- 
trated, one  volume.  Large  Square  Octavo,  Uniform  with 
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tiff"  Complete  descriptive  circular  free  upon  application. 
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gery.    Based  upon  the  above  and  Uniform  with   "Gould's 
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Corners,  $1.00;  with  Thumb  Index,  $1.25. 

HUGHES.  Compend  of  the  Practice  of  Medicine.  6th  Edition, 
Revised  and  Enlarged. 

Part  I.     Continued,  Eruptive,  and  Periodical  Fevers,  Disease 
of  the  Stomach,  Intestines,  Peritoneum,  Biliary  Passages, 
Liver,  Kidneys,  etc.,  and  General  Diseases,  etc. 
Part   II.     Diseases  of  the   Respiratory   System,   Circulatory 
System,  and  Nervous  System ;  Diseases  of  the  Blood,  etc. 

Price  of  each  part,  .80;  Interleaved,  $1.00 
Physician's  Edition.     In  one  volume,  including  the  above  two 
parts,  a  Section  on  Skin  Diseases,  and  an  Index.     6th  Re- 
vised Edition.     625  pp.     Full  Morocco,  Gilt  Edge,  $2.25 
TAYLOR.     Practice  of  Medicine.     6th  Edition.  $4.00 

TYSON.     The  Practice   of  Medicine.     By  JAMES   TTSON,   M.a , 
Professor  of  Medicine  in  the  University  of  Pennsylvania. 
Complete   Systematic  Text-book,  with   Special  Reference   to 
Diagnosis  and  Treatment.     3d  Edition,  Enlarged  and  Revised. 
Colored  Plates  and  124  other  Illustrations. 

Cloth,  $5.50 ;  Leather,  $6.50 

STOMACH.     INTESTINES. 

FENWICK.     Cancer  of  the  Stomach.     Just  Ready.  $3.00 

HEMMETER.  Diseases  of  the  Stomach.  Their  Special  Pathol- 
ogy, Diagnosis,  and  Treatment.  With  Sections  on  Anatomy, 
Analysis  of  Stomach  Contents,  Dietetics.  Surgery  of  the  Stom- 
ach, etc.  3d  Edition,  Revised.  With  15  Plates  and  41  other 
Illustrations,  a  number  of  which  are  in  color*. 

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testinal Contents,  Secretions,  Feces  and  Urine,  Intestinal 
Bacteria  and  Parasites,  Surgery  of  the  Intestines,  Dietetics, 
Diseases  of  the  Rectum,  etc.  With  Full-page  Colored  Plates 
and  many  other  Original  Illustrations.  2  volumes.  Octavo. 
Price  of  each  volume,  Cloth,  $5.00 ;  Sheep,  $6.00 

SKIN. 

BULKLEY.     The  Skin  in  Health  and  Disease.    Illustrated.     .40 
CROCKER.     Diseases  of  the  Skin.     Their  Description,  Pathol- 
ogy, Diagnosis,  and  Treatment,  with  Special  Reference  to  the 
Skin  Eruptions  of  Children.     3d  Edition,  Thoroughly  Revised. 
With  New  Illus.     Jiist  Ready.         Cloth,  $5.00;  Sheep,  $6.00 
MacLEOD.     The   Pathology  of  the  Skin.     Colored    and    other 
Illustrations.     Just  Ready.  $5.00 


J4  SUBJECT  CATALOGUE. 


SCHAMBERG.  Diseases  of  the  Skin.  3d  Edition,  Revised  and 
Enlarged.  106  Illustrations.  Being  No.  16  ?  Quii-Compend? 
Series.  Just  Ready.  Cloth,  .80;  Interleaved,  $1. 00 

VAN  HARLINGEN.  On  Skin  Diseases.  A  Practical  Manual 
of  Diagnosis  and  Treatment,  with  Special  Reference  to  Differ- 
ential Diagnosis.  3d  Edition,  Revised  and  Enlarged.  With 
Formulae  and  60  Illustrations,  some  of  which  are  printed  in 
colors.  $2.75 

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BUTLIN.  Operative  Surgery  of  Malignant  Disease.  2d  Edi- 
tion. Illustrated.  Octavo.  $4.50 

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DAVIS.  Bandaging.  Its  Principles  and  Practice.  163  Original 
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HAMILTON.     Lecturei  on  Tumors.     3d  Edition.  $1.25 

HEATH.  Minor  Surgery  and  Bandaging.  12th  Edition,  Re- 
vised and  Enlarged.  195  Illus.,  Formulae,  Diet  List,  etc.  $1.50 

HEATH.     Clinical  Lectures  on  Surgical  Subjects.     Second  Series. 

$2.00 

HORWITZ.  Compend  of  Surgery  and  Bandaging.  Including 
Minor  Surgery,  Amputations,  Fractures,  Dislocations,  Surgical 
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Edition,  very  much  Enlarged  and  Rearranged.  167  Illus.,  98 
Formulae.  Cloth,  .80;  Interleaved,  $1.00 

JACOBSON.  Operations  of  Surgery.  4th  Ed.,  Enlarged.  550 
Illus.  Two  volumes.  Cloth,  $10.00;  Leather,  $12.00 

KEAY.     Medical  Treatment  of  Gall-Stones.  $1.25 

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SBTMOUR,  M.D.  $2.50 

MAKINS.  Surgical  Experiences  in  South  Africa.  1899-1900. 
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MAYLARD.  Surgery  of  the  Alimentary  Canal.  97  Illustrations. 
2d  Edition,  Revised.  $3.00 


MEDICAL  BOOKS.  25 

MOULLIN.  Text-Book  of  Surgery.  With  Special  Reference  to 
Treatment.  3d  American  Edition.  Revised  and  edited  by 
JOHN  B.  HAMILTON,  M.D.,  LL.D.,  Professor  of  the  Principles  of 
Surgery  and  Clinical  Surgery,  Rush  Medical  College,  Chicago. 
623  Illustrations,  many  of  which  are  printed  in  colors. 

Cloth,  $6.00;  Leather,  $7.00 

SMITH.  Abdominal  Surgery.  Being  a  Systematic  Description 
of  all  the  Principal  Operations.  224  Illustrations.  6th  Edi- 
tion. 2  volumeg.  Cloth,  $10.00 

VOSWINKEL.  Surgical  Nursing.  Second  Edition,  Revised  and 
Enlarged.  Ill  Illustrations.  $1.00 

WALSHAM.  Manual  of  Practical  Surgery.  7th  Ed.,  Revised 
and  Enlarged.  483  Engravings.  950  pages.  $3.50 

TEMPERATURE  CHARTS,  ETC. 

GRIFFITH.  Graphic  Clinical  Chart  for  Recording  Tempera- 
ture, Respiration,  Pulse,  Day  of  Disease,  Date,  Age,  Sex,  Occu- 
pation, Name,  etc.  Printed  in  three  colors.  Sample  copies 
free.  Put  up  in  loose  packages  of  fifty,  50  cts.  Price  to 
Hospitals,  500  copies,  $4.00;  1000  copies,  $7.50. 

KEEN'S  Clinical  Charts.  Seven  Outline  Drawings  of  the  Body, 
on  which  may  be  marked  the  Course  of  Disease,  Fractures, 
Operations,  etc.  Each  Drawing  may  be  had  separately, 
twenty-five  to  pad,  25  cents. 

THROAT  AND  NOSE  (see  also  Ear). 

COHEN.  The  Throat  and  Voice.  Illustrated.  .40 

HALL.  Diseases  of  the  Nose  and  Throat.  2d  Edition,  Enlarged. 

Two  Colored  Plates  and  80  Illustrations.  $2.75 

HOLLOPETER.  Hay  Fever.  Its  Successful  Treatment.  $1.00 
KNIGHT.  Diseases  of  the  Throat.  A  Manual  for  Students. 

Illustrated. 


KYLE  (J.  J.).  Diseases  of  the  Ear,  Nose,  and  Throat.  A  Com- 
pend  for  Students.  Illustrated.  .80;  Interleaved,  $1.00 

McBRIDE.  Diseases  of  the  Throat,  Nose,  and  Ear.  With  Col- 
ored Illustrations  from  Original  Drawings.  3d  Ed.  $7.00 

POTTER.  Speech  and  its  Defects.  Considered  Physiologically, 
Pathologically,  and  Remedially.  $1.00 

URINE  AND  URINARY  ORGANS. 

ACTON.  The  Functions  and  Disorders  of  the  Reproductive 
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Considered  in  their  Physiological,  Social,  and  Moral  Relations. 
8th  Edition.  $1.75 

CASPER  AND  RICHTER.    Functional  Kidney  Diagnosis.    $1.50 

HOLLAND.  The  Urine,  the  Gastric  Contents,  the  Common 
Poisons,  and  the  Milk.  Memoranda,  Chemical  and  Micro- 
scopical, for  Laboratory  Use.  Illustrated  and  Interleaved. 
6th  Edition.  $1.00 

KLEEN.     Diabetes  and  Glycosuria.  $2.50 


20  SUBJECT  CATALOGUE. 

MEMMINGER.  Diagnosis  by  the  Urine.  2d  Edition.  24  Illus- 
trations. $1.00 

MORRIS.  Renal  Surgery,  with  Special  Reference  to  Stone  in  the 
Kidney  and  Ureter  and  to  the  Surgical  Treatment  of  Calculous 
Anuria.  Illustrated.  $2.00 

MOULLIN.  Enlargement  of  the  Prostate.  Its  Treatment  and 
Radical  Cure.  2d  Edition.  Illustrated.  $1.75 

MOULLIN.  Inflammation  of  the  Bladder  and  Urinary  Fever. 
Octavo.  $1.50 

SCOTT.  The  Urine.  Its  Clinical  and  Microscopical  Examina- 
tion. 41  Lithographic  Plates  and  other  Illustrations.  Quarto. 

Cloth,  $5.00 

TYSON.  Guide  to  Examination  of  the  Urine.  For  the  Use  of 
Physicians  and  Students.  With  Colored  Plate  and  Numerous 
Illustrations  engraved  on  wood.  10th  Edition,  Revised,  En- 
larged, and  partly  Rewritten.  With  New  Illustrations.  Just 
Ready.  $1.50 

VAN  NUYS.     Chemical  Analysis  of  Urine.     39  Illus.  $1.00 

VENEREAL  DISEASES. 

GOWERS.     Syphilis  and  the  Nervdus  System.  $1.00 

STURGIS  AND  CABOT.     Student's  Manual  of  Venereal  Diseases. 

7th  Revised  and  Enlarged  Edition.     12mo.  $1.25 

VETERINARY. 

BALLOU.  Equine  Anatomy  and  Physiology.  29  Graphic 
Illustrations.  .80;  Interleaved,  $1.00 


JACOBSON.  The  Operations  of  Surgery.  By 
W.  H.  A.  JACOBSON,  F.R.C.S.,  Surgeon  to 
Guy's  Hospital ;  Consulting  Surgeon  Royal 
Hospital  for  Children  and  Women  ;  and  F. 
J.  STEWARD,  F.R.C.S.,  Assistant  Surgeon 
Guy's  Hospital.  Fourth  Edition — Revised, 
Enlarged,  and  Improved.  550  Illustrations, 
Two  Volumes,  Octavo,  1524  pages. 

Cloth,  $10.00;  Sheep,  $12.00 

"  The  important  anatomical  points  are  clearly  set  forth,  the 
conditions  indicating  or  contraindicating  operative  interference 
are  given,  the  details  of  the  operations  themselves  are  brought 
forward  prominently,  and  frequently  the  after-treatment  is 
considered.  Herein  is  one  of  the  strong  points  of  the  book." — 
New  York  Medical  Journal. 


"We  know  of  no  series  of  books  issued  by  any  house  that  so 
fully  meets  our  approval  as  these  ?  Quiz-Compends?.  They  are 
well  arranged,  full  and  concise,  and  are  really  the  best  line  of 
text-books  that  could  be  found  for  either  student  or  practitioner." 
— Southern  Clinic. 

BLAKISTON'S  ?  QUIZ-COMPENDS? 

The  Best  Series  of  Manuals  for  the  Use  of  Students. 
Price  of  each,  Cloth,  .80.  Interleaved,  for  taking  Notes,  Si.oo 
These  Compends  are  based  on  the  most  popular  text-books 
and  the  lectures  of  prominent  professors,  and  are  kept  constantly 
revised,  so  that  they  may  thoroughly  represent  the  present  state 
of  the  subjects  upon  which  they  treat.  The  authors  have  had 
large  experience  as  Quiz-Masters  and  attaches  of  colleges,  and  are 
well  acquainted  with  the  wants  of  students.  They  are  arranged 
in  the  most  approved  form,  thorough  and  concise,  containing 
nearly  1000  illustrations  and  lithograph  plates,  inserted  wherever 
they  could  be  used  to  advantage.  Can  be  used  by  students  of 
any  college.  They  contain  information  nowhere  else  collected  in 
such  a  condensed,  practical  shape. 

No.  i.  POTTER.  HUMAN  ANATOMY.  Seventh  Edition.  138 
Illustrations  and  16  Platea  of  Nerves  and  Arteries. 

No.  2.  HUGHES.  PRACTICE  OF  MEDICINE.  Part  I.  Sixth 
Edition,  Enlarged  and  Improved. 

No.  3.  HUGHES.  PRACTICE  OF  MEDICINE.  Part  II.  Sixth 
Edition,  Revised  and  Improved. 

No.  4.  BRUBAKER.     PHYSIOLOGY.     Eleventh  Edition.    Illus. 

No.  5.  LANDIS.     OBSTETRICS.     Seventh    Edition.     52    Illus. 

No.  6.  POTTER.  MATERIA  MEDICA,  THERAPEUTICS,  AND 
PRESCRIPTION  WRITING.  Sixth  Revised  Edition. 

No.  7.  WELLS.     GYNECOLOGY.     Third  Edition.     140  Illus. 

No.  8.  GOULD  AND  PYLE.  DISEASES  OF  THE  EYE.  Second 
Edition.  Refraction,  Treatment,  Surgery,  etc.  109  Illus. 

No.  9.  HORWITZ.  SURGERY.  Including  Minor  Surgery, 
Bandaging,  Surgical  Diseases,  Differential  Diagnosis  and 
Treatment.  Fifth  Edition.  With  98  Formulae  and  71  Illus- 
trations. 

No.  10.  LEFFMANN.  MEDICAL  CHEMISTRY.  Fourth  Edi- 
tion. Including  Urinalysis,  Animal  Chemistry,  Chemistry 
of  Milk,  Blood,  Tissues,  the  Secretions,  etc. 

No.  ii.  STEWART.  PHARMACY.  Fifth  Edition.  Based  upon 
Prof.  Remington's  Text-Book  of  Pharmacy. 

No.  12.  BALLOU.  EQUINE  ANATOMY  AND  PHYSIOLOGY. 
29  graphic  Illustrations. 

No.  13.  WARREN.  DENTAL  PATHOLOGY  AND  DENTAL 
MEDICINE.  Third  Edition,  Illustrated. 

No.  14.  HATFIELD.     DISEASES  OF  CHILDREN.     3d  Edition. 

No.  15.  THAYER.     GENERAL  PATHOLOGY.     78  Illus. 

No.     1 6.  SCHAMBERG.     DISEASES    OF    THE    SKIN.     Third 
Edition,  Revised  and  Enlarged.     106  Illustrations. 

No.  17.  CUSHING.     HISTOLOGY.     Illustrated.  In  Frets. 

No.   18.  THAYER.     SPECIAL  PATHOLOGY.     34  Illustrations. 

No.  19.  KYLE.  DISEASES  OF  THE  EAR,  NOSE,  AND 
THROAT.  Illustrated. 

27 


DA  COSTA 


Clinical   Hematology 


A  Practical  Guide  to  the  Examination  of  the  Blood  by 
Clinical  Methods.  With  Reference  to  the  Diagnosis  of 
Disease.  With  Colored  Illustrations.  Cloth,  $5.00 

*.£*  A  new,  thorough,  systematic,  and  comprehensive 
work,  its  purpose  being,  first,  to  show  how  to  examine  the 
blood,  and  second,  how  to  diagnose  from  such  examination 
diseases  of  the  blood  itself  and  general  diseases.  The 
author's  aim  has  been  to  cover  not  alone  the  field  of  original 
research,  but  to  supply  a  book  for  the  student,  the  hospital 
physician  and  the  general  practitioner.  It  will  be  found 
wanting  in  none  of  these  respects. 

OERTEL 


Medical   Microscopy 

JUST  READY 

A  GUIDE  TO  DIAGNOSIS,  ELEMEN- 
TARY LABORATORY  METHODS, 
AND  MICROSCOPIC  TECHNIC 


By  T.   E.   OERTEL,   M.D., 

Professor  of  Pathology  and   Clinical  Microscopy,  Medical  Depart- 
ment, University  of  Georgia. 

WITH  131  ILLUSTRATIONS.    121110.   Cloth,  $2.00 


The  Pocket  Cyclopedia,  of 
Medicine   and   Surgery 

Full  Limp  Leather,  Round  Corners,  Gilt  Edges,  $1.00 
With  Thumb  Index,  $1.25 

Uniform  ivith  "Gould's  Pocket  Dictionary" 


A  concise  practical  volume  of  nearly  600 
pages,  containing  a  vast  amount  of  infor- 
mation on  all  medical  subjects,  including 
Diagnosis  and  Treatment  of  Disease, 
with  Formulas  and  Prescriptions,  Emer- 
gencies, Poisons,  Drugs  and  Their  Uses, 
Nursing,  Surgical  Procedures,  Dose  List 
in  both  English  and  Metric  Systems,  etc. 

By  Drs.  Gould  and  Pyle 

Based  upon  their  large  "Cyclopedia  of 
Medicine  and  Surgery."  <£  &  <£ 


*^*  This  is  a  new  book  which  will  prove  of  the  greatest 
value  to  students.  It  is  to  the  broad  field  of  general  medi- 
cal information  what  "Gould's  Pocket  Dictionary"  is  to 
the  more  special  one  of  definition  and  pronunciation  of 
words.  The  articles  are  concise  but  thorough,  and  arranged 
in  shape  for  quick  reference.  In  no  other  book  can  be 
found  so  much  exact  detailed  knowledge  so  conveniently 
classified,  so  evenly  distributed,  so  methodically  grouped. 
It  is  Multum  in  Parvo.  Sample  Pages  Free. 
29 


A  NEW  EDITION 


CROCKER  ON  THE  SKIN 


The  Diseases  of  the  Skin.  Their  Description,  Pathology, 
Diagnosis,  and  Treatment,  with  Special  Reference  to  the 
Skin  Eruptions  of  Children.  By  H.  RADCLIFFE  CROCKER, 
M.D.,  Physician  to  the  Department  of  Skin  Diseases,  Uni- 
versity College  Hospital,  London.  With  new  Illustrations. 

Third  Edition,  Rewritten  and  Enlarged 

OCTAVO.    JUST  READY;   CLOTH,  $5.00 

*#*  This  new  edition  will  easily  hold  the  high  position 
given  the  previous  printings.  The  author  is  a  member  of 
American,  English,  French,  German,  and  Italian  Dermato- 
logical  Societies,  and  a  recognized  authority  the  world  over. 


STURGIS— MANUAL  OF 
VENEREAL  DISEASES 


By  F.  R.  STURGIS,  M.D.,  Sometime  Clinical  Professor  of 
Venereal  Diseases  in  the  Medical  Department  of  the  Uni- 
versity of  the  City  of  New  York.  Seventh  Edition,  Revised 
and  in  Part  Rewritten  by  the  Author  and  FOLLEN  CABOT, 
M.D.,  Instructor  in  Genito-Urinary  and  Venereal  Diseases 
in  the  Cornell  University  Medical  College.  I2mo.  216 
pages.  Cloth,  $1.25 

*x*  This  manual  was  originally  written  for  students' 
use,  and  is  as  concise  and  as  practical  as  possible.  It  pre- 
sents a  careful,  condensed  description  of  the  commoner 
forms  of  venereal  diseases  which  occur  in  the  practice  of 
the  general  physician,  together  with  the  most  approved 
remedies. 

80 


FOR  THE^  DISSECTING  ROOM 

Holden's  Anatomy — Seventh  Edition 
320  Illustrations 

A  Manual  of  the  Dissections  of  the  Human  Body.  By  JOHN 
LANGTON,  F.R.C.S.  Carefully  Revised  by  A.  HEWSON,  M.D., 
Demonstrator  of  Anatomy,  Jefferson  Medical  College,  Phila- 
delphia, etc.  320  Illustrations.  Two  small  compact  vol- 
umes. I2mo. 

Vol.  I.  Scalp,  Face,  Orbit,  Neck,  Throat,  Thorax,  Upper 
Extremity.  435  pages.  153  Illustrations. 

Oil  Cloth,  $1.50 

Vol.  II.  Abdomen,  Perineum,  Lower  Extremity,  Brain, 
Eye,  Ear,  Mammary  Gland,  Scrotum,  Testes. 
445  Pages.  167  Illustrations. 

Oil  Cloth,  $1.50 

Each  volume  sold  separately. 


Hughes    a.i\d    Keith  —  Dissections 
Illustrated 

A  Manual  of  Dissections  by  ALFRED  W.  HUGHES,  M.B., 
M.R.C.s.  (Edin. ),  late  Professor  of  Anatomy  and  Dean  of 
Medical  Faculty,  King's  College,  London,  etc.,  and  ARTHUR 
KEITH,  M.D.,  Joint  Lecturer  on  Anatomy,  London  Hospital 
Medical  College,  etc.  In  three  parts.  With  527  Colored 
and  other  Illustrations. 

I.     Upper  and  Lower  Extremity.     38  Plates,  116  other 
Illustrations.  Cloth,  $3.00 

II.      Abdomen.      Thorax.     4    Plates,    149    other   Illus- 
trations. Cloth,  $3.00 
III.      Head,   Neck,  and  Central   Nervous   System.       16 
Plates,  204  other  Illustrations.           Cloth,  $3.00 

Each  volume  sold  separately. 

*.£*  The  student  will  find  it  of  great  advantage  to  have 
a    "  Dissector "    to    supplement   his   regular   text-book   on 
anatomy.     These  books  meet  all  requirements,  and  as  they 
can  be  purchased  in  parts  as  wanted,  the  outlay  is  small. 
31 


EDGAR'S 

OBSTETRICS 

A    NEW    TEXT -BOOK 
I  22  i    Illustrations 


Edgar's  Obstetrics  excels  all 
other  works  on  this  subject 
in  completeness,  in  uni- 
formity and  consistency  in 
arrangement,  thoroughness 
and  clearness  in  handling 
details,  and  in  the  number 
and  usefulness  of  its  illus- 
trations. 

OCTAVO.      CM.oni,    56.oo;    SIIKKP,    <7.oo 


OC! 


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